LSR receptor, its activity, its cloning, and its applications to the diagnosis, prevention and/or treatment of obesity and related risks or complications

ABSTRACT

The present invention relates to a new complex receptor polypeptide LSR (Lipolysis Stimulated Receptor), characterized by its functional activities, the cloning of the cDNAs complementary to the messenger RNAs encoding each of the subunits of the multimeric complex, vectors and transformed cells, methods of diagnosis and of selection of compounds which can be used as medicament for the prevention and/or treatment of pathologies and/or of pathogeneses such as obesity and anorexia, hyperlipidemias, atherosclerosis, diabetes, hypertension, and more generally the various pathologies associated with abnormalities in the metabolism of cytokines.

INTRODUCTION

The present invention relates to a new complex receptor polypeptide LSR(Lipolysis Stimulated Receptor), characterized by its functionalactivities, the cloning of the cDNAs complementary to the messenger RNAsencoding each of the subunits of the multimeric complex, vectors andtransformed cells, methods of diagnosis and of selection of compoundswhich can be used as medicament for the prevention and/or treatment ofpathologies and/or of pathogeneses such as obesity and anorexia,hyperlipidemias, atherosclerosis, diabetes, hypertension, and moregenerally the various pathologies associated with abnormalities in themetabolism of cytokines.

Obesity is a public health problem which is both serious and widespread:in industrialized countries, a third of the population has an excessweight of at least 20% relative to the ideal weight. The phenomenoncontinues to worsen, in regions of the globe whose economies are beingmodernized, such as the Pacific islands, and in general. In the UnitedStates, the number of obese people has passed from 25% at the end of the70s to 33% at the beginning of the 90s.

Obesity considerably increases the risk of developing cardiovascular ormetabolic diseases. It is estimated that if the entire population had anideal weight, the risk of coronary insufficiency would decrease by 25%and that of cardiac insufficiency and of cerebral vascular accidents by35%. Coronary insufficiency, atheromatous disease and cardiacinsufficiency are at the forefront of the cardiovascular complicationsinduced by obesity. For an excess weight greater than 30%, the incidenceof coronary diseases is doubled in subjects under 50 years. Studiescarried out for other diseases are equally eloquent. For an excessweight of 20%, the risk of high blood pressure is doubled. For an excessweight of 30%, the risk of developing a non-insulin-dependent diabetesis tripled. That of hyperlipidemias is multiplied by 6.

The list of diseases whose onset is promoted by obesity is long:hyperuricemia (11.4% in obese subjects, against 3.4% in the generalpopulation), digestive pathologies, abnormalities in hepatic functions,and even certain cancers.

Whether the physiological changes in obesity are characterized by anincrease in the number of adipose cells, or by an increase in thequantity of triglycerides stored in each adipose cell, or by both, thisexcess weight results mainly from an imbalance between the quantities ofcalories consumed and those of the calories used by the body. Studies onthe causes of this imbalance have been in several directions. Some havefocused on studying the mechanism of absorption of foods, and thereforethe molecules which control food intake and the feeling of satiety.Other studies have been related to the basal metabolism, that is to saythe manner in which the body uses the calories consumed.

The treatments for obesity which have been proposed are of four types.Food restriction is the most frequently used. The obese individuals areadvised to change their dietary habits so as to consume fewer calories.This type of treatment is effective in the short-term. However, therecidivation rate is very high. The increase in calorie use throughphysical exercise is also proposed. This treatment is ineffective whenapplied alone, but it improves, however, weight loss in subjects on alow-calorie diet. Gastrointestinal surgery, which reduces the absorptionof the calories ingested, is effective but has been virtually abandonedbecause of the side effects which it causes. The medicinal approach useseither the anorexigenic action of molecules involved at the level of thecentral nervous system, or the effect of molecules which increase energyuse by increasing the production of heat. The prototypes of this type ofmolecule are the thyroid hormones which uncouple oxidativephosphorylations of the mitochondrial respiratory chain. The sideeffects and the toxicity of this type of treatment make their usedangerous. An approach which aims to reduce the absorption of dietarylipids by sequestering them in the lumen of the digestive tube is alsoin place. However, it induces physiological imbalances which aredifficult to tolerate: deficiency in the absorption of fat-solublevitamins, flatulence and steatorrhoea. Whatever the envisagedtherapeutic approach, the treatments of obesity are all characterized byan extremely high recidivation rate.

The molecular mechanisms responsible for obesity in humans are complexand involve genetic and environmental factors. Because of the lowefficiency of the treatments known up until now, it is urgent to definethe genetic mechanisms which determine obesity, so as to be able todevelop better targeted medicaments.

More than 20 genes have been studied as possible candidates, eitherbecause they have been implicated in diseases of which obesity is one ofthe clinical manifestations, or because they are homologues of genesinvolved in obesity in animal models. Situated in the 7q31 chromosomalregion, the OB gene is one of the most widely studied. Its product,leptin, is involved in the mechanisms of satiety. Leptin is a plasmaprotein of 16 kDa produced by the adipocytes under the action of variousstimuli. Obese mice of the ob/ob type exhibit a deficiency in the leptingene; this protein is undetectable in the plasma of these animals. Theadministration of leptin obtained by genetic engineering to ob/ob micecorrects their relative hyperphagia and allows normalization of theirweight. This anorexigenic effect of leptin calls into play a receptor ofthe central nervous system: the ob receptor which belongs to the familyof class 1 cytokine receptors. The ob receptor is deficient in obesemice of the db/db strain. The administration of leptin to these mice hasno effect on their food intake and does not allow substantial reductionin their weight. The mechanisms by which the ob receptors transmit thesignal for satiety are not precisely known. It is possible thatneuropeptide Y is involved in this signalling pathway. It is importantto specify at this stage that the ob receptors are not the onlyregulators of appetite. The Melanocortin 4 receptor is also involvedsince mice made deficient in this receptor are obese (Gura, 1997).

The discovery of leptin and the characterization of the leptin receptorat the level of the central nervous system have opened a new route forthe search for medicaments against obesity. This model, however, rapidlyproved disappointing. Indeed, with only one exception (Montague et al.,1997), the genes encoding leptin or its ob receptor have proved to benormal in obese human subjects. Furthermore and paradoxically, theplasma concentrations of leptin, the satiety hormone, are abnormallyhigh in most obese human subjects. Most of the therapeutic researchefforts in this direction have centred on the characterization of theeffect of leptin at the level of the central nervous system.

SUMMARY OF THE INVENTION

The present invention results from a focusing of the research effort onthe discovery of the mechanisms of leptin elimination. The most widelyaccepted working hypothesis is that the plasma levels of leptin are highin obese subjects because this hormone is produced by the adipose tissueand that the fatty mass is increased in obese subjects. The inventorshave formulated a different hypothesis and have postulated that theconcentrations of leptin are increased in obese individuals because theclearance of this hormone is reduced. This deficiency causes a leptinresistance syndrome and the obese individual develops a suitableresponse to the high concentrations of leptin. In this perspective, thetreatment of obese subjects ought to consist not in an increase in theleptin levels but in a normalization thereof. At this stage, it isessential to recall that the ob type receptors are signalling typereceptors. These receptors can bind leptin at the level of the plasmamembrane but cannot cause the protein to enter inside the cell for it tobe degraded therein. The ob receptors are not endocytosis receptors.

LSR Receptor

The inventors have characterized a receptor, in particular hepatic,called LSR receptor, whose activity is dual. The LSR receptor allows, onthe one hand, endocytosis of lipoproteins, when it is activated by thefree fatty acids, thus serving as a pathway for the clearance oflipoproteins. This pathway serves mainly, but not exclusively, for theclearance of particles high in triglycerides of intestinal origin (Mannet al., 1995). This activity, expressed most particularly at the hepaticlevel, is dependent on the presence of free fatty acids which, bybinding to the receptor, induce a reversible change in the conformationof this complex and allow it to bind, with a high affinity, variousclasses of lipoproteins such as those containing apoprotein B orapoprotein E.

On the other hand, under normal conditions, in the absence of free fattyacids, the complex receptor LSR does not bind lipoproteins, but iscapable of binding a cytokine, in particular leptin, and then ofinternalizing it and of degrading it.

The present invention therefore relates to a purified LSR receptor, inparticular of hepatic cells, characterized in that it is capable, in thepresence of free fatty acids, of binding lipoproteins, and in theabsence of free fatty acids, of binding a cytokine, preferably leptin.

According to the invention, this LSR receptor is, in addition,characterized in that the bound lipoproteins or the bound cytokine areincorporated into the cell and then degraded, the bound lipoproteinscontaining in particular apoprotein B or E.

It should be understood that the invention does not relate to the LSRreceptors in a natural form, that is to say that they are not taken intheir natural environment but obtained by purification from naturalsources, or alternatively obtained by genetic recombination, oralternatively by chemical synthesis and capable, in this case, ofcontaining non-natural amino acids, as will be described below. Theproduction of a recombinant LSR receptor, which may be carried out usingone of the nucleotide sequences according to the invention, isparticularly advantageous because it makes it possible to obtain anincreased level of purity of the receptor.

More particularly, the invention relates to a purified rat LSR receptor,characterized in that it comprises at least one subunit having amolecular weight of about 66 kDa and a subunit having a molecular weightof about 58 kDa.

Preferably, the purified rat LSR receptor of the present invention ischaracterized in that it contains an a subunit comprising the amino acidsequence of SEQ ID 2 or a sequence homologous thereto, or an α′ subunitcomprising the amino acid sequence of SEQ ID 4 or a sequence homologousthereto, and one, preferably three, β subunits comprising the amino acidsequence of SEQ ID 6 or a sequence homologous thereto.

The invention also relates to a purified mouse LSR receptor,characterized in that it comprises at least one subunit having amolecular weight of about 66 kDa and a subunit having a molecular weightof about 58 kDa.

Preferably, the purified mouse LSR receptor of the present invention ischaracterized in that it contains an a subunit comprising the amino acidsequence of SEQ ID 16 or a sequence homologous thereto, or an α′ subunitcomprising the amino acid sequence of SEQ ID 17 or a sequence homologousthereto, and one, preferably three, β subunits comprising the amino acidsequence of SEQ ID 18 or a sequence homologous thereto.

The invention also relates to a purified human LSR receptor,characterized in that it comprises at least one subunit having amolecular weight of about 72 kDa and a subunit having a molecular weightof about 64 kDa.

Preferably, the purified human LSR receptor of the present invention ischaracterized in that it contains an α′ subunit comprising the aminoacid sequence of SEQ ID 8 or a sequence homologous thereto, or an α′subunit comprising the amino acid sequence of SEQ ID 10 or a sequencehomologous thereto, and one, preferably three, β subunits comprising theamino acid sequence of SEQ ID 12 or a sequence homologous thereto.

A particularly preferred embodiment of the LSR receptors of the presentinvention is a recombinant LSR receptor obtained by expressing, in arecombinant host, one or more nucleotide sequences according to theinvention. This preferred recombinant receptor consists of an α or α′subunit and one, preferably three, β subunits, in particular an α or α′subunit and three β subunits of a human LSR receptor.

Polypeptide Sequences of LSR

The invention relates to polypeptides, characterized in that they are aconstituent of an LSR receptor according to the invention.

It should be understood that the invention does not relate to thepolypeptides in a natural form, that is to say that they are not takenin their natural environment. Indeed, the invention relates to thepeptides obtained by purification from natural sources, or alternativelyobtained by genetic recombination, or alternatively by chemicalsynthesis, and capable, in this case, of containing non-natural aminoacids, as will be described below. The production of a recombinantpolypeptide, which may be carried out using one of the nucleotidesequences according to the invention or a fragment of one of thesesequences, is particularly advantageous because it makes it possible toobtain an increased level of purity of the desired polypeptide.

The invention therefore relates to a purified, isolated or recombinantpolypeptide comprising a sequence of at least 5, preferably at least 10to 15, consecutive amino acids of an LSR receptor, as well as thehomologues, equivalents or variants of the said polypeptide, or one oftheir fragments. Preferably, the sequence of at least 10 to 15 aminoacids of the LSR receptor is a biologically active fragment of an LSRreceptor.

More particularly, the invention relates to purified, isolated orrecombinant polypeptides comprising a sequence of at least 10 to 15amino acids of a rat LSR receptor, of a mouse LSR receptor or of a humanLSR receptor.

In the present description, the term polypeptide will be used to alsodesignate a protein or a peptide.

Nucleotide Sequences of LSR

The subject of the present invention is also purified nucleic acidsequences, characterized in that they encode an LSR receptor or apolypeptide according to the invention.

The invention relates to a purified nucleic acid, characterized in thatit comprises at least 8, preferably at least 10 and more particularly atleast 15 consecutive nucleotides of the polynucleotide of a genomic,cDNA or RNA sequence of the LSR receptor, as well as the nucleic acidsequences complementary to this nucleic acid. More particularly, theinvention relates to the purified, isolated or recombinant nucleic acidscomprising a sequence of at least 8, preferably at least 10 and moreparticularly at least 15 consecutive nucleotides of the polynucleotideof a nucleic sequence of a mouse LSR receptor or of a human LSRreceptor.

The invention also relates to the variant, mutated, equivalent orhomologous nucleic sequences of the nucleic sequences according to theinvention, or one of their fragments. It finally relates to thesequences capable of hybridizing specifically with the nucleic sequencesaccording to the invention.

The invention therefore also relates to the nucleic acid sequencescontained in the gene encoding the LSR receptor, in particular each ofthe exons of the said gene or a combination of exons of the said gene,or alternatively a polynucleotide extending over a portion of one ormore exons. Preferably, these nucleic acids encode one or morebiologically active fragments of the human LSR receptor.

The present invention also relates to the purified nucleic acidsequences encoding one or more elements for regulating the expression ofthe LSR gene. Also included in the invention are the nucleic acidsequences of the promoter and/or regulator of the gene encoding thereceptor according to the invention, or one of their allelic variants,the mutated, equivalent or homologous sequences, or one of theirfragments.

The invention also relates to the purified nucleic sequences forhybridization comprising at least 8 nucleotides, characterized in thatthey can hybridize specifically with a nucleic sequence according to theinvention.

Preferably, nucleic acid fragments or oligonucleotides, having assequences the nucleotide sequences according to the invention can beused as probes or primers.

The invention also comprises methods for screening cDNA and genomic DNAlibraries, for the cloning of the isolated cDNAs and/or the genes codingfor the receptor according to the invention, and for their promotersand/or regulators, characterized in that they use a nucleic sequenceaccording to the invention.

The nucleic sequences, characterized in that they are capable of beingobtained by one of the preceding methods according to the invention orthe sequences capable of hybridizing with the said sequences, form partof the invention.

Vectors, Host Cells and Transgenic Animals

The invention also comprises the cloning and/or expression vectorscontaining a nucleic acid sequence according to the invention.

The vectors according to the invention, characterized in that theycomprise elements allowing the expression and/or the secretion of thesaid sequences in a host cell, also form part of the invention.

The invention comprises, in addition, the host cells, in particular theeukaryotic and prokaryotic cells, transformed with the vectors accordingto the invention, as well as the mammals, except man, comprising one ofthe said transformed cells according to the invention.

Among the mammals according to the invention, there will be preferredanimals such as mice, rats or rabbits, expressing a polypeptideaccording to the invention, the phenotype corresponding to the normal orvariant LSR receptor, in particular mutated of human origin.

These cells and animals can be used in a method of producing arecombinant polypeptide according to the invention and can also serve asa model for analysis and screening.

The invention also relates to the use of a cell, of a mammal or of apolypeptide according to the invention for studying the expression andthe activity of the receptor according to the invention, and the director indirect interactions between the said receptor and chemical orbiochemical compounds which may be involved in the activity of the saidreceptor.

The invention also relates to the use of a cell, of a mammal or of apolypeptide according to the invention for screening a chemical orbiochemical compound capable of interacting directly or indirectly withthe receptor according to the invention, and/or capable of modulatingthe expression or the activity of the said receptor.

Production of Polypeptides Derived From the LSR Receptor

The invention also relates to the synthesis of synthetic or recombinantpolypeptides of the invention, in particular by chemical synthesis orusing a nucleic acid sequence according to the invention.

The polypeptides obtained by chemical synthesis and capable ofcomprising non-natural amino acids corresponding to the said recombinantpolypeptides are also included in the invention.

The method of producing a polypeptide of the invention in recombinantform is itself included in the present invention, and is characterizedin that the transformed cells are cultured under conditions allowing theexpression of a recombinant polypeptide having a polypeptide sequenceaccording to the invention, and in that the said recombinant polypeptideis recovered.

The recombinant polypeptides, characterized in that they are capable ofbeing obtained by the said method of production, also form part of theinvention.

Antibodies

The mono- or polyclonal antibodies or fragments thereof, chimeric orimmunoconjugated antibodies, characterized in that they are capable ofspecifically recognizing a polypeptide or a receptor according to theinvention, form part of the invention.

There may be noted in particular the advantage of antibodiesspecifically recognizing certain polypeptides, variants or fragments,which are in particular biologically active, according to the invention.

The invention also relates to methods for the detection and/orpurification of a polypeptide according to the invention, characterizedin that they use an antibody according to the invention.

The invention comprises, in addition, purified polypeptides,characterized in that they are obtained by a method according to theinvention.

Moreover, in addition to their use for the purification of polypeptides,the antibodies of the invention, in particular the monoclonalantibodies, may also be used for the detection of these polypeptides ina biological sample.

More generally, the antibodies of the invention may be advantageouslyused in any situation where the expression, normal or abnormal, of apolypeptide of the LSR receptor, normal or mutated, needs to beobserved.

Detection of Allelic Variability and Diagnosis

Also forming part of the invention are the methods for the determinationof an allelic variability, a mutation, a deletion, a loss ofheterozygosity or a genetic abnormality, characterized in that they usea nucleic acid sequence or an antibody according to the invention.

These methods relate to, for example, the methods for the diagnosis ofthe predisposition to obesity, to the associated risks, or topathologies associated with abnormalities in the metabolism ofcytokines, by determining, in a biological sample from the patient, thepresence of mutations in at least one of the sequences described above.The nucleic acid sequences analysed may be either the genomic DNA, thecDNA or the mRNA.

Nucleic acids or antibodies based on the present invention can also beused to allow a positive and differential diagnosis in a patient takenin isolation, or a pre-symptomatic diagnosis in an at risk subject, inparticular with a familial history.

In addition, the detection of a specific mutation may allow an evolutivediagnosis, in particular as regards the intensity of the pathology orthe probable period of its appearance.

Screening of Compounds of Interest

Also included in the invention are the methods for selecting chemical orbiochemical compounds capable of interacting, directly or indirectly,with the receptor or the polypeptide or nucleotide sequences accordingto the invention, and/or allowing the expression or the activity of theLSR receptor to be modulated.

The invention relates in particular to a method for selecting chemicalor biochemical compounds capable of interacting with a nucleic acidsequence contained in a gene encoding an LSR receptor, the said methodbeing characterized in that it comprises bringing a host cell expressingan LSR receptor or a fragment of the said receptor into contact with acandidate compound capable of modifying the expression or the regulationof the expression of the said nucleic sequence, and detecting, directlyor indirectly, a modification of the expression or of the activity ofthe LSR receptor.

The invention also relates to a method for selecting chemical orbiochemical compounds capable of interacting with the LSR receptor, thesaid method being characterized in that it comprises bringing an LSRreceptor or a fragment of the said receptor, or a host cell expressingan LSR receptor or a fragment of the said receptor, into contact with acandidate compound capable of modifying the LSR activity, and detecting,directly or indirectly, a modification of the activity of the LSRreceptor or the formation of a complex between the candidate compoundand the said LSR receptor or the said polypeptide.

The invention comprises the compounds capable of interacting directly orindirectly with an LSR receptor as well as the compounds capable ofinteracting with one or more nucleic sequences of the LSR receptor. Italso comprises the chemical or biochemical compounds allowing theexpression or the activity of the receptor according to the invention tobe modulated. The compounds, characterized in that they were selected byone of the methods according to the present invention, also form part ofthe invention.

In particular, among these compounds according to the invention, thereare preferred the antibodies according to the invention, thepolypeptides according to the invention, the nucleic acids,oligonucleotides and vectors according to the invention, or a leptin orone of its derived compounds, preferably one of its protein variants, orleptins which are chemically modified or are obtained by geneticrecombination, or the protein gC1qR or one of its analogues, or one oftheir fragments.

The invention comprises, finally, compounds capable of modulating theexpression or the activity of the receptor according to the invention,as medicament for the prevention of pathologies and/or of pathogenesessuch as obesity and anorexia, hyperlipidemias, atherosclerosis,diabetes, hypertension, and more generally the various pathologiesassociated with abnormalities in the metabolism of cytokines.

DETAILED DESCRIPTION

The LSR Receptor

The invention relates to a purified LSR receptor (“Lipolysis StimulatedReceptor”), preferably hepatic, consisting of at least one α or α′subunit and at least one β subunit. The α subunit has a molecular weightof about 66 kDa in rats and in mice and of about 72 kDa in humans. Theα′ subunit has a molecular weight of about 64 kDa in rats and in miceand of about 70 kDa in humans. The β subunit has a molecular weight ofabout 58 kDa in rats and in mice and of about 64 kDa in humans.

The inventors have formulated the hypothesis according to which the mostabundant, and probably the most active, form of the LSR receptor is thatin which an α or α′ subunit and three β subunits exist. It appears,however, possible that the α and α′ subunits, on the one hand, and the βsubunit, on the other, have distinct biological functions and that thesefunctions can be performed in a cell independently of their assembly inthe form of a receptor.

The inventors have also observed that a complex can form between the LSRreceptor and the gC1qR receptor having a molecular weight of about 33kDa, or a homologous protein. It appears that the gC1qR receptor istransiently combined with the LSR receptor and that the presence of aC1q protein or of homologous proteins makes it possible not only todissociate gC1qR from the LSR receptor but also to activate the LSRreceptor, including in the absence of fatty acids.

Activity of the LSR Receptor and Applications

The present invention therefore relates to a receptor, in particular ofhepatic cells, characterized in that it is capable, in the presence offree fatty acids, of binding lipoproteins, and in the absence of freefatty acids, of binding a cytokine, preferably the bound leptin,lipoproteins and cytokine being incorporated and then degraded by thecell, it being possible for the said receptor, in addition, to bind thegC1qR protein or one of its analogous proteins.

Clearance of Lipoproteins

The LSR receptor represents the principal pathway for the elimination oflipoproteins of intestinal origin and of particles high intriglycerides, in particular VLDLs and chylomicrons. The LSR receptorcan also serve as a pathway for the elimination of LDLs, particles highin cholesterol, which are for the most part removed by the LDL receptorpathway, but of which about 30% are eliminated at the hepatic level bypathways different from the LDL receptor.

The inventors have in fact demonstrated that the LSR receptor is capableof binding lipoproteins, in particular the lipoproteins high intriglycerides, and then of internalizing and degrading them. Thislipoprotein clearance activity by the receptor requires the presence offree fatty acids, for example oleate, and is inhibited in the presenceof antibodies directed against LSR or against peptides derived from LSR.

Clearance of Cytokines

The inventors have also demonstrated that in the absence of free fattyacids, for example oleate, the LSR receptor is capable of bindingcytokines, preferably leptin. The leptin clearance function is, however,only possible if the receptor has not bound fatty acids produced by thehepatic lipase or by the hormone-sensitive lipase of the adipose tissue.Once the cytokines have been bound, the LSR receptor internalizes themand degrades them. This cytokine, preferably leptin, degradationactivity is inhibited by antibodies directed against LSR or againstpeptides derived from LSR.

The inventors have shown that it is the α subunit of the LSR receptorwhich is most particularly involved in the binding of cytokines, andpreferably of leptin.

Furthermore, the inventors have shown, with the aid of mice, that, invivo, the LSR receptors carry out the hepatic capturing of cytokines,preferably of leptin.

The high levels of leptin in all obese human subjects can be explainedby several molecular mechanisms which are capable of reducing thehepatic clearance of leptin, including in particular:

-   a) alteration of one or more genes for LSR, and/or of their    promoters;-   b) facilitation, by post-transcriptional modifications, of the    allosteric rearrangement allowing the passage from the    cytokine-competent conformation to the lipoprotein receptor    conformation;-   c) deficiency in the transport of vesicles containing LSR from, or    towards, the plasma membrane (this function depends on the integrity    of the cytoskeleton);-   d) increase in the degradation of LSR;-   e) increase in the lipid calorie ration which, by diverting the    receptor towards the clearance of lipoproteins, reduces in part its    capacity to degrade leptin.    Control of LSR Activity by the Cytokines

Finally, the inventors have demonstrated that cytokines, preferablyleptin, modulate the activity of the LSR receptor in the presence offree fatty acids. More particularly, the cytokines increase thelipoprotein clearance activity of the LSR receptor and more precisely,the binding, internalization and degradation of the VLDLs and LDLs. Thisincrease in the LSR activity could be the result of the increase in theapparent number of LSR receptor at the surface of the cells following anincrease in protein synthesis and following a mobilization ofendocytosis vesicles. In addition, the inventors have shown, with theaid of mice, that, in vivo, cytokines, preferably leptin, are capable ofreducing postprandial lipaemic response.

Leptin, and probably other cytokines, are therefore regulators of theactivity of LSR. A syndrome of resistance to leptin, or to othercytokines, can lead to a hypertriglyceridemia, which is either permanentor limited to the postprandial phase.

Treatment of Obesity

The role played by LSR in the clearance of leptin makes it possible toformulate a physiopathological model which requires a revision of thestrategies used for treating obesity. It is indeed essential to reducethe concentrations of leptin in obese human subjects in order to restorethe physiological fluctuations of this hormone.

Accordingly, it is possible to envisage using compounds for thetreatment of obesity allowing modulation of the number of LSR receptors,of their recycling rate, or of the change in their conformation, and/orallowing in particular:

-   1. leptinemia, and therefore the sensations of satiety and of    hunger, to be controlled;-   2. normal leptin concentrations to be restored and normal regulation    of dietary habit by the normal perception of the sensations of    hunger and of satiety;-   3. triglyceridernia to be controlled;-   4. the plasma concentrations of residues of chylomicrons, highly    atherogenic particles, to be regulated.

The role played by the LSR receptor in the hepatic clearance oflipoproteins of intestinal region makes it possible to envisage usingcompounds capable of modulating the expression and/or the activity ofLSR in order to modulate the distribution of lipids of dietary originbetween the peripheral tissues, in particualr the adipose tissues, andthe liver. A treatment of obesity will consist in promoting the hepaticdegradation of lipoproteins, and thereby reducing their storage in theadipose tissue, and regulating their plasma concentrations. The lattereffect makes it possible to envisage the use of such compounds to reducethe risks associated with obesity, in particular the atherogenic risks.

Treatments of Anorexia and of Cachexia

It is possible to envisage using methods of regulating the activities ofLSR to introduce treatments which make it possibile to overcome thevicious circle which characterizes anorexia nervosa. By reducing thenumber of receptors, it should be possible to promote weight gain inanorexic or undernourished subjects.

Under these conditions, it is advantageous to selectively inhibit theclearance of leptin by using synthetic peptides or pharmacologicalmolecules which either reduce the synthesis of LSR or block its capacityto bind leptin and/or lipoproteins, or alternatively increase thecatabolism of the receptor.

Treatment of Abnormalities in the Metabolism of Cytokines

Analysis of the primary structure of the α subunit of LSR, as describedbelow, shows a site homologous to the cytokine binding sites present ontheir receptors, as well as two routing signals which allow endocytosisand rapid degradation of ligands in the lysozomes. This observation isnew in the sense that the cytokine receptors do not allow theinternalization and the degradation of ligands. These receptors havebeen characterized on the basis of their intracellular signallingproperties.

Thus, in addition to it having the property of allowing the proteolyticdegradation of lipoproteins and of leptin, it is highly probable thatthe LSR receptor also carries out the degradation of other cytokines.This function can be studied by virtue of the anti-LSR antibodies and oftransfected CHO cells expressing the α subunit of LSR as described inExample 4. The involvement of LSR in the clearance of cytokines isessential because these molecules play an important role in theregulation of the metabolism of lipids, of the metabolism of glucose,and in the regulation of food intake and of weight gain.

The molecular mechanisms by which the cytokines modulate thephysiological functions involved in obesity and its complications arenumerous and complex. It is worth noting, however, the fact thatabnormalities in the metabolism of cytokines are associated withhypertriglyceridemia which frequently accompanies viral, bacterial orprotozoal infections. Moreover, cytokines, and more particularly TumorNecrosis Factor (TNF), induce a transient hypertriglyceridemia similarto that observed in certain forms of obesity-related diabetes.

The reduction in the number of LSR receptors expressed in the liver ofobese mice could explain a deficiency in the elimination of somecytokines, this deficiency causing metabolic disruptions such as thosefound in obesity. The use of hepatic cells in culture, and of thevarious models of obese animals cited below, will make it possible todetermine, among all the cytokines and more particularly those whichinduce weight loss (IL-6, LIF, OSM, CNTF, IL-11, IL-12α, as well as TNFαand TNFβ), those which modulate the expression and/or the activity ofLSR. The determination of such cytokines can, for example, be carriedout using methods such as those presented in Examples 4 to 6.

Finally, analysis of the primary structure of the α LSR revealspotential phosphorylation sites. This opens the perspective of aregulation of cellular activity by the LSR receptor. A particularlyimportant example would be the involvement of LSR in the regulation ofthe production of “Acute Phase Proteins” under the impetus of variousstimuli, including cytokines.

The involvement of LSR in the clearance and the degradation of cytokinesmay, in addition, not be limited to the liver. Indeed, while it has beendemonstrated that the expression of LSR is predominantly hepatic, it isalso certain that the expression of this receptor is not limited to thisorgan. Preliminary Northern-blot analysis on various human tissues hasbeen able to reveal, in addition to the hepatic products, expressionproducts in the kidney and in the testicle. A more thorough analysiswill make it possible to show the different tissues expressing LSR inhumans. In this perspective, LSR could be involved in the degradation ofcytokines not only at the hepatic level, but also at the level of theperipheral tissues. A deficiency in this activity could be involved inthe pathogenesis of autoimmune diseases, of multiple sclerosis and ofrheumatoid arthritis. Accumulation of cytokines is frequently found inthe pathogenesis of these diseases.

Polypeptide Sequences of the LSR Receptor

The invention relates to polypeptides, characterized in that they are aconstituent of an LSR receptor according to the invention. The inventionrelates more particularly to the polypeptides characterized in that theyconstitute the α, α′ or β subunits of the LSR receptor.

The invention relates more particularly to a purified, isolated orrecombinant polypeptide comprising a sequence of at least 5, preferablyof at least 10 to 15 consecutive amino acids of an LSR receptor, as wellas the homologues, equivalents or variants of the said polypeptide, orone of their fragments. Preferably, the sequence of at least 10 to 15amino acids of the LSR receptor is a biologically active fragment of anLSR receptor.

Preferably, the invention relates to purified, isolated or recombinantpolypeptides comprising a sequence of at least 10 to 15 amino acids of arat LSR receptor, of a mouse LSR receptor or of a human LSR receptor.

In a first preferred embodiment of the invention, the polypeptide ischaracterized in that it comprises a sequence of at least 10 to 15consecutive amino acids of a sequence chosen from the group comprisingthe sequences of SEQ ID 2, SEQ ID 4 and SEQ ID 6, as well as thevariants, equivalents or homologues of this polypeptide, or one of theirfragments. Preferably, the polypeptide is a homologue or a biologicallyactive fragment of one of the abovementioned sequences.

In a second preferred embodiment of the invention, the polypeptide ischaracterized in that it comprises a sequence of at least 10 to 15consecutive amino acids of a sequence chosen from the group comprisingthe sequences of SEQ ID 16, SEQ ID 17 and SEQ ID 18, as well as thevariants, equivalents or homologues of this polypeptide, or one of theirfragments. Preferably, the polypeptide is a homologue or a biologicallyactive fragment of one of the abovementioned sequences.

In a third preferred embodiment of the invention, the polypeptide ischaracterized in that it comprises a sequence of at least 10 to 15consecutive amino acids of a sequence chosen from the group comprisingthe sequences of SEQ ID 8, SEQ ID 10 and SEQ ID 12, as well as thevariants, equivalents or homologues of this polypeptide, or one of theirfragments. Preferably, the polypeptide is a homologue or a biologicallyactive fragment of one of the abovementioned sequences.

Among the preferred polypeptides of the invention, there will be notedparticularly the polypeptides having the human sequence SEQ ID 8, SEQ ID10 or SEQ ID 12, as well as those having the rat sequence SEQ ID 2, SEQID 4 or SEQ ID 6, or those having the mouse sequence SEQ ID 16, SEQ ID17 or SEQ ID 18. The fragments corresponding to the domains representedin FIGS. 1 to 6, whose positions on the sequences corresponding to SEQID 2, 8 or 16, are indicated in Tables 1, 3 and 4.

Finally, the invention also relates to the polypeptides of SEQ ID 29 andSEQ ID 30.

The present invention also relates to polypeptides comprising thepolypeptides described above, as well as their homologous, equivalent orvariant polypeptides, as well as the fragments, preferably biologicallyactive, of the said polypeptides.

Among the polypeptides according to the invention, also preferred arethe polypeptides comprising or consisting of an amino acid sequencechosen from the amino acid sequences as described above, characterizedin that the said polypeptides are a constituent of the receptoraccording to the invention.

Analysis of the Polypeptide Sequences of the α, α′ and β Subunits of theLSR Receptor

The systematic analysis of the products of the 3 rat cDNAs described inthe present application is schematically represented in FIG. 1. The αsubunit of the rat LSR receptor, a protein encoded by the longer cDNA(LSR-Rn-2097), has the following characteristics.

Potential glycosylation sites are found at positions 12-14 and 577-579.A potential site of attachment of glycosaminoglycans is found atposition 14-17.

Several phosphorylation sites are located at the level of theNH₂-terminal end (positions 193-196, 597-600, 169-171, 172-174, 401-403,424-426, 464466, 467-469, 185-188, 222-225, 436-439, 396-399, 504-507,530-533, 624-627, 608-615), suggesting that the latter is orientedtowards the intracellular region.

Moreover, the protein has, on the NH₂-terminal side, a hydrophobic aminoacid sequence separated into two parts by 2 amino acids inducing ahairpin structure in which the two arms would consist of hydrophobicamino acids. It is reasonable to assume that this region represents thefatty acid binding site of LSR. The glove-finger structure thus producedcan accommodate an aliphatic hydrocarbon chain. The two amino acids are,more precisely in the case of rat LSR, two Prolines situated atpositions 31 and 33 of the polypeptide sequence of the α subunit.

Still on the NH₂-terminal side is a consensus sequence for binding toclathrin, a protein which lawns the inner surface of the “coated pits”(Chen et al., 1990). These specific regions of the plasma membrane allowrapid endocytosis of membrane proteins. Such a consensus sequence isfound at the level of the LRP-α₂-macroglobulin receptor, of CRAM and ofthe LDL receptor (Herz et al., 1988; Lee et al., 1990; Goldstein et al.,1995). The consequence of a mutation at this level is a substantialdelay in the internalization of the LDLs and induces familialhypercholesterolemia (Davis et al., 1986).

The receptor then possesses a hydrophobic amino acid sequence whichconstitutes a potential transmembrane domain. The length of this segmentallows only one passage across the phospholipid bilayer (Brendel et al.,1992).

Between this clathrin binding signal and the hydrophobic chaincorresponding to the single transmembrane segment are 2 motifs LI et LL(Letourneur et al., 1992). These two motifs are found in the followingproteins: glut 4 glucose carrier (Verhey et al., 1994); the nonvariantchain and the histocompatibility complex class 11 (Zhong et al., 1997Parra-Lopez et al., 1997). These signals control endocytosis and theintracellular addressing of proteins in the peripheral membrane system.

On the C-terminal side, there is then a cysteine-rich region whichexhibits homology with the cytokine receptors and more particularly: theTNF 1 and 2 (Tumor Necrosis Factor 1 and 2) receptors; the low-affinityNGF (Nerve Growth factor) receptor; the Shope fibroma virus TNF solublereceptor; CD40, CD27 and CD30, receptors for the cytokines CD40L, CD27Land CD30L; the T cell protein 4-1BB, receptor for the putative cytokine4-1BBL, the FAS antigen (APO 1), receptor for the FASL protein involvedin apoptosis, the T cell 0X40 antigen, receptor for the cytokine 0X40L,and the vaccinia virus A53 protein (Cytokines and their receptors, 1996;Banner et al., 1993).

In addition to this cysteine-rich segment, there is a region of aminoacids which are alternately charged + and − (Brendel et al., 1992). Thisregion provides a potential binding site for the apoprotein ligands ApoB and Apo E.

This region contains, in addition, an RSRS motif found in lamin (Simoset al., 1994) and in SF2′ (Krainer et al., 1991).

The LSR α′ form encoded by the LSR-Rn-2040 cDNA possesses all thedomains described above based on the LSR α sequence encoded by theLSR-Rn-2097 cDNA, with the exception of the LI/LL element, whose Leucinedoublet is removed by alternative splicing. Although possessingsequences which are very similar, the subunits α encoded by LSR-Rn-2097and α′ encoded by LSR-Rn-2040 could therefore differ in their recyclingrate and their addressing. The β form encoded by LSR-Rn-1893 does notpossess a transmembrane domain or a region rich in cysteines andhomologous to the cytokine receptors. However, it possesses at theNH₂-terminal level the hydrophobic region separated by a repetition ofprolines, the region rich in charged amino acids and the RSRS motif.This constituent is probably positioned entirely outside the cell whereit is bound via disulphide bridges either to the product of LSR-Rn-2040,or to that of LSR-Rn-2097.

Table 1 below lists the different domains or motifs described above,indicates whether or not they belong to each of the subunits of the LSRreceptor, as well as the positions of the start and end of the saiddomains or motifs, or of regions carrying the said domains or motifs, asindicated in the sequence of SEQ ID 2.

TABLE 1 Position on SEQ ID 2 Presence on: Domain or motif Start End α α′β Potential fatty acid binding site 23 41 X X X Potential clathrinbinding site 104 107 X X X Signal for transport: LI 183 184 X X X LL 195196 X Transmembrane domain 204 213 X X Potential cytokine receptor site214 249 X X RSRS motif 470 473 X X X Potential lipoprotein ligandbinding site 544 557 X X XComparison of the Polypeptide Sequences of the LSR Receptors in Rats,Mice and Humans

The lengths of the polypeptide sequences, as well as the SEQ IDs oftheir respective sequences in the listing included, of the three typesof subunit of the LSR receptors according to the invention, in rats,mice and humans, are indicated in Table 2a below.

TABLE 2a Polypeptide Rat Mouse Human α subunit 593 aa (SEQ ID 2) 594 aa(SEQ ID 16) 649 aa (SEQ ID 8) α′ subunit 574 aa (SEQ ID 4) 575 aa (SEQID 17) 630 aa (SEQ ID 10) β subunit 525 aa (SEQ ID 6) 526 aa (SEQ ID 18)581 aa (SEQ ID 12)

These polypeptide sequences were obtained from each of the threecorresponding cDNA sequences, in rats, mice and humans, which will bedescribed in detail later. These polypeptide sequences were obtainedfrom each of the three corresponding cDNA sequences, in rats, mice andhumans, which will be described in detail later. The nomenclature usedto designate these cDNA sequences, which reflects their length in termsof nucleotides, as well as the SEQ IDs of their respective sequences inthe listing included, are presented in Table 2b below.

TABLE 2b cDNAc Rat Mouse Human α subunit LSR-Rn-2097 LSR-Mm-1886LSR-Hs-2062 (SEQ ID 1) (SEQ ID 13) (SEQ ID 7) α′ subunit LSR-Rn-2040LSR-Mm-1829 LSR-Hs-2005 (SEQ ID 3) (SEQ ID 14) (SEQ ID 9) β subunitLSR-Rn-1893 LSR-Mm-1682 LSR-Hs-1858 (SEQ ID 5) (SEQ ID 15) (SEQ ID 11)

The protein sequence, corresponding to the α subunit of the LSRreceptor, deduced from the LSR-Hs-2062 sequence has a length of 649amino acids. It is aligned with the protein sequences deduced fromLSR-Mm-1886, 594 amino acids long, and from LSR-Rn-2097, 593 amino acidslong (FIGS. 2A and 2B). The conservation of the protein sequences isvery high (respectively 80.2% and 82.2% identity for 591 and 590overlapping amino acids). The functional domains identified in theprotein sequence of the rat LSR α are found in the human LSR α sequenceas well as in that of the murine LSR α (FIGS. 2A and 2B).

The human proteins corresponding to the LSR-Hs-2005 (α′) and LSR-Hs-1858(β) forms have a predicted size of 630 and 581 amino acids respectively.The rat proteins corresponding to the LSR-Rn-2040 (α′) and LSR-Rn-1893(β) forms have a predicted size of 574 and 525 amino acids respectively.The mouse proteins corresponding to the LSR-Mm-1829 (α′) and LSR-Mm-1682(β) forms have a predicted size of 575 and 526 amino acids respectively.The alignment of the three human forms (FIGS. 3A and 3B), of the threeforms described in rats (FIGS. 4A and 4B) and of the three formsdescribed in mice (FIGS. 5A and 5B) shows that in the three species, allthe protein forms conserve the NPGY signal for binding to clathrin andthe RSRS motif. The human (product of LSR-Hs-2062), rat (product ofLSR-Rn-2097) and mouse (product of LSR-Mm-1886) long forms (α) exhibitall the functional characteristics of LSR. The three short forms (β)(respective products of LSR-Hs-1817, LSR-Rn-1893 and LSR-Mm-1682) losethe di-leucine domain for lysosomal addressing, the transmembrane domainand the cytokine receptor signature. It is also possible to observe thatthe three intermediate forms (α′) (product of LSR-Hs-2005, ofLSR-Rn-2040 and LSR-Mn-1829) lose the di-leucin domain, thetransmembrane domain and the domain corresponding to the cytokinereceptor signature being conserved (FIGS. 3A, 3B, 4A, 4B, 5A and 5B).FIG. 6 finally represents the proteins derived from the three cDNA formsidentified in humans, and the motifs carried by each of them as a resultof the splicing from which each is derived.

Table 3 below lists the different domains or motifs described above, aswell as the positions of the start and end of the said domains ormotifs, or of regions carrying the said domains or motifs, as indicatedin the mouse SEQ ID 16 sequence.

TABLE 3 Position on SEQ ID 16 Presence on: Domain or motif Start End αα′ β Potential fatty acid binding site 23 41 X X X Potential clathrinbinding site 104 107 X X X Signal for transport: LI 183 184 X X X LL 195196 X Transmembrane domain 204 213 X X Potential cytokine receptor site214 249 X X RSRS motif 470 473 X X X Potential lipoprotein ligandbinding site 544 558 X X X

Table 4 below lists the different domains or motifs described above, aswell as the positions of the start and end of the said domains ormotifs, or of regions carrying the said domains or motifs, as indicatedin the human SEQ ID 8 sequence.

TABLE 4 Position on SEQ ID 8 Presence on: Domain or motif Start End α α′β Potential fatty acid binding site 76 94 X X X Potential clathrinbinding site 157 160 X X X Signal for transport: LI 236 237 X X X LL 248249 X Transmembrane domain 257 266 X X Potential cytokine receptor site267 302 X X RSRS motif 527 530 X X X Potential lipoprotein ligandbinding site 601 613 X X X

In conclusion, the similarity in the sequence and structure of LSR whichis described above makes it possible to extrapolate to humans theobservations made in rats and/or mice.

Homologous polypeptide will be understood to mean the polypeptidesexhibiting, compared with the natural polypeptide, certain modificationssuch as in particular a deletion, truncation, extension, chimeric fusionand/or mutation, in particular a point mutation. Among the homologouspolypeptides, those in which the amino acid sequence exhibits at least80%, preferably 90%, homology with the amino acid sequences of thepolypeptides according to the invention are preferred.

Equivalent polypeptide will be understood to mean a polypeptide havingat least one of the activities of the LSR receptor, in particular theactivity of the receptor for lipoproteins or chylomicrons, the activityof the receptor for cytokine, in particular leptin, or the activity ofthe receptor for the gC1q-R protein or one of its analogous proteins.Equivalent polypeptide will also be understood to mean any polypeptideresulting from the alternative splicing of the genomic nucleic sequenceencoding the polypeptides according to the invention.

Variant polypeptide (or protein variant) will be understood to mean allthe mutated polypeptides which may exist, in particular in human beings,and which correspond in particular to truncations, deletions and/oradditions of amino acid residues, substitutions or mutations, inparticular point mutations, as well as the artificial variantpolypeptides which will nevertheless be called variant polypeptides. Inthe present case, the variant polypeptides will be in particular partlyassociated with the onset and with the development of obesity oranorexia. They may also be associated with the onset and/or developmentof the risks or complications associated with obesity, in particular atthe cardiovascular level, and/or of pathologies associated withabnormalities in the metabolism of cytokines.

Polypeptide fragment is understood to mean a polypeptide or a peptideencoded by a nucleic sequence comprising a minimum of 15 nucleotides orbases, preferably 20 bases or 30 bases. These fragments may comprise inparticular a point mutation, compared with the normal polypeptidesequence, or may correspond to specific amino acid sequences of variantpolypeptides, artificial or existing in humans, such as those linked toa polymorphism linked in particular to obesity or to the abovementionedpathologies.

Biologically active fragment will be understood to mean in particular afragment of an amino acid sequence of a polypeptide:

-   -   exhibiting at least one of the LSR receptor activities, in        particular the lipoprotein receptor activity, or the cytokine,        particularly leptin, receptor activity and/or cell signalling        activity, and/or    -   capable of being recognized by an antibody specific for the        receptor according to the invention, and/or    -   capable of being recognized by a compound capable, for example        by neutralizing the binding of a ligand specific for the said        receptor, of modulating the activity of the LSR receptor, and/or    -   capable of modulating the addressing and/or cellular location of        the LSR receptor, and/or    -   more generally constituting a biologically active domain or        motif of the LSR receptor.        Among the preferred biologically active fragments according to        the invention, there are in particular:    -   the fragments comprising a clathrin binding site,    -   the fragments comprising a fatty acid binding site, in        particular a fatty acid binding site comprising a hydrophobic        amino acid sequence separated into two parts by two contiguous        prolines, which induce a hairpin structure whose arms consist of        hydrophobic amino acids,    -   the fragments comprising a hydrophobic region constituting a        transmembrane domain,    -   the fragments comprising a region capable of controlling        endocytosis and intracellular addressing of the proteins in the        peripheral membrane system, in particular a fragment comprising        a site containing the LI and LL motifs,    -   the fragments comprising a cytokine binding site, in particular        a site including a cysteine-rich region,    -   the fragments comprising a region defining a potential binding        site for lipoprotein ligands such as ApoB and ApoE, in        particular a region comprising a sequence of amino acids        alternately charged + and −, and    -   the fragments comprising an RSRS motif.

There are in particular among these fragments polypeptides as defined inTables 1, 2 and 4, or any fragments of the nucleotides of SEQ ID 2, 8 or16, comprising the said polypeptides, and any equivalent, homologous orvariant fragments.

Other preferred fragments include antigenic peptides such as thosehaving the sequences SEQ ID 29 and 30.

Nucleotide Sequences of the LSR Receptor

The subject of the present invention is isolated nucleic acid sequences,characterized in that they encode an LSR receptor or a polypeptideaccording to the invention.

More particularly, the invention relates to a purified nucleic acid,characterized in that it comprises at least 8, preferably at least 10and more particularly at least 15 consecutive nucleotides of thepolynucleotide of SEQ ID 19, as well as the nucleic acid sequencescomplementary to this nucleic acid.

The invention also relates to a purified nucleic acid, characterized inthat it comprises at least 8, preferably at least 10 and moreparticularly at least 15 consecutive nucleotides of the polynucleotideof SEQ ID 41, as well as the nucleic acid sequences complementary tothis nucleic acid.

The invention also relates to a purified nucleic acid encoding the humanLSR receptor, characterized in that it comprises a nucleotide sequencecorresponding to nucleotides 1898 to 21094, particularly to nucleotides2001 to 20979, more particularly to nucleotides 2145 to 20979 of SEQ ID19, as well as the nucleic acid sequences complementary to this nucleicacid.

The invention also relates to the nucleic acid sequences contained inthe gene encoding the human LSR receptor, in particular each of theexons of the said gene or a combination of exons of the said gene, oralternatively a polynucleotide extending over a portion of one or moreexons. Preferably, these nucleic acids encode one or more biologicallyactive fragments of the human LSR receptor.

The invention also relates to a purified nucleic acid, characterized inthat it comprises a nucleotide sequence corresponding to nucleotides 1to 1897 of SEQ ID 19, as well as the nucleic acid sequencescomplementary to this nucleic acid.

The invention also relates to a purified nucleic acid, characterized inthat it comprises a nucleotide sequence corresponding to nucleotides21095 to 22976 of SEQ ID 19, as well as the nucleic acid sequencescomplementary to this nucleic acid.

The invention also relates to a purified nucleic acid, characterized inthat it comprises a nucleotide sequence chosen from the group comprisingthe sequences of SEQ ID 7, SEQ ID 9 and SEQ ID 11, as well as thenucleic acid sequences complementary to this nucleic acid.

The invention also relates to a purified nucleic acid, characterized inthat it comprises a nucleotide sequence chosen from the group comprisingthe sequences of SEQ ID 1, SEQ ID 3 and SEQ ID 5, as well as the nucleicacid sequences complementary to this nucleic acid.

The invention also relates to a purified nucleic acid, characterized inthat it comprises a nucleotide sequence chosen from the group comprisingthe sequences of SEQ ID 13, SEQ ID 14 and SEQ ID 15, as well as thenucleic acid sequences complementary to this nucleic acid.

The invention also relates to a purified nucleic acid, characterized inthat it comprises a nucleotide sequence corresponding to nucleotides1898 to 2001 of SEQ ID 19 or preferably to nucleotides 1898 to 2144 ofSEQ ID 19, as well as the nucleic acid sequences complementary to thisnucleic acid.

The invention also relates to a purified nucleic acid, characterized inthat it comprises a nucleotide sequence corresponding to nucleotides20980 to 21094 of SEQ ID 19, as well as the nucleic acid sequencescomplementary to this nucleic acid.

Among the nucleic acids according to the invention, the nucleic acidshaving the nucleotide sequence chosen from the group comprising thesequences of SEQ ID 7, SEQ ID 9 and SEQ ID 11, the sequences of SEQ ID1, SEQ ID 3 and SEQ ID 5, as well as the sequences of SEQ ID 13, SEQ ID14 and SEQ ID 15, as well as their complementary sequences, arepreferred.

Also forming part of the invention are the variant, mutated, equivalentor homologous sequences of the sequences according to the invention, aswell as their fragments and the nucleic sequences capable of hybridizingspecifically with the sequences according to the invention.

Human Genomic Sequence

The invention therefore relates to the genomic sequence of the human LSRreceptor, preferably the sequence of SEQ ID 19, as well as theircomplementary sequences or one of their allelic variants, the mutated,equivalent or homologous sequences, or one of their fragments.

The gene for human LSR (SEQ ID 19) comprises 10 exons distributed over21 094 bp. The size of the exons is respectively: 356, 345, 120, 57,147, 174, 60, 132, 626 and 141 bp (Table 5).

TABLE 5 EXON START END 5′ SPLIC. BL 5′ 3′ SPLIC. BL 3′ Ex1 1898 2253 — —GTACGG +2 Ex2 3437 3781 CAG +1 GTATGT +1 Ex3 12067 12186 CAG +2 GTGAGT+1 Ex4 15047 15103 CAG +2 GTACGG +1 Ex5 15668 15814 CAG +2 GTAAGT +1 Ex619481 19654 CAG +2 GTGAGG +1 Ex7 19801 19860 CAG +2 GTGAGA +1 Ex8 1995820089 TAG +2 GTAAGC +1 Ex9 20231 20856 CAG +2 GTGAGG   0 Ex10 2094621094 CAG   0 —

The EXON column indicates the exons numbered from 1 to 10 in the 5′-3′order of their position on the genomic sequence. The START and ENDcolumns indicate respectively the position of the first and of the lastnucleotide of the exon considered. The sequences of the splicing sitebordering the exon in 5′ and 3′ are indicated in the 5′SPLIC and 3′SPLICcolumns. The BL 5′ and BL 3′ columns indicate the number of bases in 5′and in 3′, respectively, of an exon which will be used in the readingframe of the messenger only after splicing. For example: as exon 7 has afree base in 3′, this exon can be joined to the 5′ end of exon 8 whichhas 2 free bases in 5′. The combination 1 base+2 bases constitutes thecodon which was destroyed by the intron in the genomic sequence. Exon 7may be joined by its 3′ end to any exon having two free bases in 5′; ifthe new codon created does not correspond to a stop codon, the openreading frame will be conserved.

Exons 1 and 2 as well as 9 and 10 are necessarily co-spliced, thusforming a 5′ block corresponding to exons 1 and 2 and a 3′ blockcorresponding to exons 9 and 10. The functional minimal messenger,corresponding to the product of these four exons, could therefore have asize of about 1 331 bp. For the other exons, all the possiblecombinations make it possible to conserve the open reading frame.

The size of the noncoding exons in 5′ could not be determined withprecision. Indeed, the rat 5′ UTR sequences are too divergent from thoseof humans to finalize the analysis of these sequences and to identifythe real 5′ end of the human LSR cDNA. This can be carried out byisolating the 5′ end of the human LSR messengers by the 5′ end capturemethods developed by the inventors (WO 96/34981). The polyadenylationsite described below is the only one which is present before the USF2gene, situated in 3′ of the human LSR gene. It is therefore very likelythat the untranslated 3′ region of this gene is very short (of anestimated size of about 100 bp). All the sizes given in relation to thehuman LSR cDNA molecules will therefore have to be adjusted according tothe size of the untranslated 5′ end. The human cDNA sequence obtainedtaking into account all the exons deduced from the analysis of thegenomic sequence have a size of 2 158 bp. This form could correspond tothe LSR-Rn-2097 form.

The location of some of the signals for expression of the nucleotidesequence of SEQ ID 19 is presented in Table 6 which follows.

TABLE 6 Signal Start End preferred ATG 2145 2147 other possible ATG 20012003 STOP 20977 20979 POLY Ad 21065 21070

The characteristic elements of the messenger RNA molecule are describedin the Signal column: Initiation of translation (ATG), termination oftranslation (STOP) and polyadenylation signal (POLY Ad). The Start andEnd columns indicate the position as nucleotide for the start and end ofthese signals on the genomic sequence SEQ ID 19. An ATG signal forinitiation of translation is preferred to another because it provides anenvironment which is more suitable for initiation.

The present invention also relates to the purified nucleic acidsequences encoding one or more elements for regulating the expression ofthe human LSR gene. Also included in the invention are the nucleic acidsequences of the promoter and/or regulator of the gene encoding thereceptor according to the invention, or one of their allelic variants,the mutated, equivalent or homologous sequences, or one of theirfragments.

The invention relates more particularly to a purified nucleic acidsituated in 5′ of the coding sequence of the LSR gene. This nucleic acidis characterized in that it comprises a nucleotide sequencecorresponding to nucleotides 1 to 1897 of SEQ ID 19, as well as thenucleic acid sequences complementary to this nucleic acid. Shorterfragments of this nucleic acid may also be used as promoters forexpression of the LSR gene or of any other sequence encoding aheterologous polypeptide.

The invention also relates to a purified nucleic acid situated in 3′ ofthe transcribed sequence of the LSR gene. This nucleic acid ischaracterized in that it comprises a nucleotide sequence correspondingto nucleotides 21095 to 22976 of SEQ ID 19, as well as the nucleic acidsequences complementary to this nucleic acid. Shorter fragments of thisnucleic acid can also be used as elements regulating the expression ofgenes.

Finally, the invention also relates to the genomic sequence of the humanLSR receptor, preferably the sequence of SEQ ID 41, as well as theircomplementary sequences, or one or their allelic variants, the mutated,equivalent or homologous sequences, or one of their fragments.

Comparison of the Genomic Organizations in Humans, Rats and Mice

It is advantageous to note that a syntheny (conservation of theorganization of certain chromosomal regions between species) between themouse chromosome 7 region where the Lisch7 gene is located, in theimmediate vicinity of USF2, and the human chromosome 19 region 19q13,carrying LSR, is well described. The organization of the two Lisch7/LSRand USF2 genes is conserved between species. Likewise, Apo E, which isof a more centromeric location relative to these genes, exists both inmice and in humans. It is remarkable that the LSR lipoprotein receptorand one of their ligands ApoE are located in the same chromosomalregion. Indeed, the receptor and the ligand are frequently co-regulated.Such a situation would make it possible to envisage that the phenomenaobserved in mice are applicable to humans.

Human, Rat and Mouse cDNA Sequences

The invention relates, in addition, to 3 different cDNAs derived fromthe LSR receptor gene by alternative splicing. These 3 cDNAs have beenidentified in humans, rats and mice (Table 2b). They encode the threetypes of LSR receptor subunits, α (long), α′ (intermediate) and β(short). The longest cDNA contains the totality of the 10 exons of thegene. The intermediate cDNA does not comprise exon 4. Finally, theshortest cDNA does not contain exons 4 and 5.

The human LSR-Hs-2062 cDNA nucleotide sequence, encoding the α subunitof the LSR receptor, and the rat LSR-Rn-2097 cDNA nucleotide sequenceare 78.6% identical over 1 955 bp which overlap. These figures arerespectively 78.8% and 1 851 bp when the murine LSR-Mm-1886 sequence(long form) is aligned with the human sequence. This reflects a veryhigh conservation of the nucleic sequences between species. The highestdivergence levels are observed in the untranslated 5′ end (when thesequence is available), in the first coding exon and in the untranslated3′ end (FIGS. 7 A, 7B, 7C, 7D and 7E).

The invention therefore also relates to a purified nucleic acid,characterized in that it is chosen from the group comprising thesequences of SEQ ID 7, SEQ ID 9 and SEQ ID 11, the sequences SEQ ID 1,SEQ ID 3 and SEQ ID 5, and the sequences SEQ ID 13, SEQ ID 14 and SEQ ID15, as well as the nucleic acid sequences complementary to this nucleicacid, or one of their allelic variants, the mutated, equivalent orhomologous sequences, or one of their fragments.

The nucleic acids constituting the coding frames of the abovementionednucleic acids, between the codons for initiation and for termination oftranslation, also form part of the invention.

The nucleic acids encoding the polypeptide fragments according to theinvention are also part of the invention. It will be particularly notedthat the nucleic acids encode the fragments described in Tables 1, 3 and4.

Thus, Table 7 describes the position of such nucleic acid fragments onthe human sequence of SEQ ID 7.

TABLE 7 Position on the cDNA of SEQ 7 Domain or motif Start EndPotential fatty acid binding site 329 385 Potential clathrin bindingsite 572 583 Signal for transport: LI 809 814 LL 845 850 Transmembranedomain 872 901 Potential cytokine receptor site 902 1009 RSRS motif 16821693 Potential lipoprotein ligand binding site 1904 1942

The invention also relates to a purified nucleic acid corresponding tothe sequence of the 5′UTR of the cDNAs encoding the human LSR receptor.This nucleic acid is characterized in that it comprises a nucleotidesequence corresponding to nucleotides 1898 to 2001 of SEQ ID 19 orpreferably to nucleotides 1898 to 2144 of SEQ ID 19, as well as thenucleic acid sequences complementary to this nucleic acid. Shorterfragments of this nucleic acid can also be used.

The invention also relates to a purified nucleic acid corresponding tothe sequence of the 3′UTR of the cDNAs encoding the LSR receptor. Thisnucleic acid is characterized in that it comprises a nucleotide sequencecorresponding to nucleotides 20980 to 21094 of SEQ ID 19, as well as thenucleic acid sequences complementary to this nucleic acid. Shorterfragments of this nucleic acid can also be used.

The invention also relates to the purified nucleic acids correspondingrespectively to the sequences of the 5′UTR or of the 3′UTR of the cDNAsencoding the rat or mouse LSR receptor. Shorter fragments of thisnucleic acid can also be used.

The 5′UTR and 3′UTR may contain elements (“responsive elements” and“enhancers”) which are involved in the regulation of transcription andof translation. These regions have in particular a role in the stabilityof the mRNAs. Furthermore, the 5′UTR comprises the Shine-Delgarno motifwhich is essential for the translation of the mRNA.

Nucleic acid, nucleic sequence or nucleic acid sequence are understoodto mean an isolated natural, or a synthetic, DNA and/or RNA fragmentcomprising, or otherwise, non-natural nucleotides, designating a precisesuccession of nucleotides, modified or otherwise, allowing a fragment, asegment or a region of a nucleic acid to be defined.

Equivalent nucleic sequences are understood to mean nucleic sequencesencoding the polypeptides according to the invention taking into accountthe degeneracy of the genetic code, the complementary DNA sequences andthe corresponding RNA sequences, as well as the nucleic sequencesencoding the equivalent polypeptides.

Homologous nucleic sequences are understood to mean the nucleicsequences encoding the homologous polypeptides and/or the nucleicsequences exhibiting a level of homology of at least 80%, preferably90%. According to the invention, the homology is only of the statisticaltype, which means that the sequences have a minimum of 80%, preferably90%, of nucleotides in common. They are preferably sequences capable ofhybridizing specifically with a sequence of the invention. Preferably,the specific hybridization conditions will be like those found in theexamples, or such that they ensure at least 95% homology.

The length of these nucleic sequences for hybridization can vary from 8,10, 15, 20 or 30 to 200 nucleotides, particularly from 20 to 50nucleotides, more particularly from 20 to 30 nucleotides.

Allele or allelic variant will be understood to mean the natural mutatedsequences corresponding to polymorphisms present in human beings and, inparticular, to polymorphisms which can lead to the onset and/or to thedevelopment of obesity or of anorexia. These polymorphisms can also leadto the onset and/or to the development of risks or complicationsassociated with obesity, in particular at the cardiovascular level,and/or of pathologies associated with abnormalities in the metabolism ofcytokines.

Mutated nucleic sequences are understood to mean the nucleic sequencescomprising at least one point mutation compared with the normalsequence.

While the sequences according to the invention are in general normalsequences, they are also mutated sequences since they comprise at leastone point mutation and preferably at most 10% of mutations compared withthe normal sequence.

Preferably, the present invention relates to mutated nucleic sequencesin which the point mutations are not silent, that is to say that theylead to a modification of the amino acid encoded in relation to thenormal sequence. Still more preferably, these mutations affect aminoacids which structure the LSR complex and/or receptor or thecorresponding domains and fragments thereof. These mutations may alsoaffect amino acids carried by the regions corresponding to the receptorsites, for lipoproteins or cytokines, in particular leptin, or to sitesfor binding of cofactors, in particular or free fatty acids, oralternatively to phosphorylation sites. These mutations may also affectthe sequences involved in the transport, addressing and membraneanchorage of LSR.

In general, the present invention relates to the normal LSRpolypeptides, the mutated LSR polypeptides as well as fragments thereofand to the corresponding DNA and RNA sequences, the LSR polypeptidesdesignating polypeptides of the receptor according to the invention.

According to the invention, the fragments of nucleic sequences may inparticular encode domains of receptors and polypeptides possessing afunction or a biological activity as defined above, contain domains orregions situated upstream or downstream of the coding sequence andcontaining elements for regulating the expression of the LSR gene oralternatively possessing a sequence allowing their use as a probe or asa primer in methods of detection, identification or amplification ofnucleic sequences. These fragments preferably have a minimum size of 8,of 10 bases, and fragments of 20 bases, and preferably of 30 bases, willbe preferred.

Among the nucleic fragments which may be of interest, in particular fordiagnosis, there should be mentioned, for example, the genomic intronsequences of the gene for the LSR complex, such as in particular thejoining sequences between the introns and the exons, normal or mutated.

The nucleic acid sequences which can be used as sense or antisenseoligonucleotides, characterized in that their sequences are chosen fromthe sequences according to the invention, also form part of theinvention.

Among the nucleic acid fragments of interest, there should thus bementioned, in particular the antisense oligonucleotides, that is to saywhose structure ensures, by hybridization with the target sequence,inhibition of the expression of the corresponding product. There shouldalso be mentioned the sense oligonucleotides which, by interaction withthe proteins involved in the regulation of the expression of thecorresponding product, will induce either inhibition, or activation ofthis expression.

The sequences carrying mutations which may be involved in the promoterand/or regulatory sequences of the genes for the LSR complex, which mayhave effects on the expression of the corresponding proteins, inparticular on their level of expression, also form part of the precedingsequences according to the invention.

The nucleic sequences which can be used as primer or probe,characterized in that their nucleic sequence is a sequence of theinvention, also form part of the invention.

The present invention relates to all the primers which may be deducedfrom the nucleotide sequences of the invention and which may make itpossible to detect the said nucleotide sequences of the invention, inparticular the mutated sequences, using in particular a method ofamplification such as the PCR method, or a related method.

The present invention relates to all the probes which may be deducedfrom the nucleotide sequences of the invention, in particular sequencescapable of hybridizing with them, and which may make it possible todetect the said nucleotide sequences of the invention, in particular todiscriminate between the normal sequences and the mutated sequences.

The invention also relates to the use of a nucleic acid sequenceaccording to the invention as a probe or a primer for the detectionand/or the amplification of a nucleic acid sequence according to theinvention.

All the probes and primers according to the invention may be labelled bymethods well known to persons skilled in the art, in order to obtain adetectable and/or quantifiable signal.

The present invention also relates to the nucleotide sequences which maycomprise non-natural nucleotides, in particular sulphur-containingnucleotides, for example, or nucleotides of α or β structure.

The present invention relates, of course, to both the DNA and RNAsequences, as well as the sequences which hybridize with them, as wellas the corresponding double-stranded DNAs.

In the text which follows, the preceding DNA sequences will be calledgenes for the LSR complex, whether they are normal or pathologicsequences.

It should be understood that the present invention does not relate tothe genomic nucleotide sequences in their natural chromosomalenvironment, that is to say in the natural state. They are sequenceswhich have been isolated, that is to say that they have been collecteddirectly or indirectly, for example by copying (cDNA), their environmenthaving been at least partially modified.

Thus, this may also be both cDNA and genomic DNA, partially modified orcarried by sequences which are at least partially different from thesequences carrying them naturally.

These sequences may also be termed non-natural.

The invention also comprises methods for screening cDNA and genomic DNAlibraries, for the cloning of the isolated cDNAs, and/or the genescoding for the receptor according to the invention, and for theirpromoters and/or regulators, characterized in that they use a nucleicsequence according to the invention. Among these methods, there may bementioned in particular:

-   -   the screening of cDNA libraries and the cloning of the isolated        cDNAs (Sambrook et al., 1989; Suggs et al., 1981; Woo et al.,        1979), with the aid of the nucleic sequences according to the        invention,    -   the screening of 5′ end tag libraries (WO 96/34981) for nucleic        sequences according to the invention, and thus the isolation of        tags allowing the cloning of complete cDNAs and the        corresponding promoters from genomic DNA libraries,    -   the screening of genomic libraries, for example of BACs,        (Chumakov et al., 1992; Chumakov et al., 1995) and, optionally,        a genetic analysis by FISH (Cherif et al., 1990) with the aid of        sequences according to the invention, allowing isolation and        chromosomal location, and then the complete sequencing of the        genes encoding the LSR receptor.

Also included in the invention is a sequence, in particular a genomicsequence encoding a receptor or a polypeptide according to theinvention, or a nucleic acid sequence of a promoter and/or regulator ofa gene encoding a receptor or a polypeptide according to the invention,or one of their allelic variants, a mutated, equivalent or homologoussequence, or one of their fragments, characterized in that it is capableof being obtained by one of the preceding methods according to theinvention, or a sequence capable of hybridizing with the said sequences.

Vectors, Host Cells and Transgenic Animals

The invention also comprises the cloning and/or expression vectorscontaining a nucleic acid sequence according to the invention.

The vectors according to the invention, characterized in that theycomprise the elements allowing the expression and/or the secretion ofthe said sequences in a host cell, also form part of the invention.

The vectors characterized in that they comprise a promoter and/orregulator sequence according to the invention, or a sequence forcellular addressing according to the invention, or one of theirfragments, also form part of the invention.

The said vectors will preferably comprise a promoter, signals forinitiation and termination of translation, as well as appropriateregions for regulation of transcription. They must be able to be stablymaintained in the cell and may optionally possess particular signalsspecifying the secretion of the translated protein.

These different control signals are chosen according to the cellularhost used. To this end, the nucleic acid sequences according to theinvention may be inserted into autonomously replicating vectors insidethe chosen host, or integrative vectors of the chosen host.

Among the autonomously replicating systems, there will be preferablyused according to the host cell, systems of the plasmid or viral type,it being possible for the viral vectors to be in particular adenoviruses(Perricaudet et al., 1992), retroviruses, poxviruses or herpesviruses(Epstein et al., 1992). Persons skilled in the art know the technologieswhich can be used for each of these systems.

When the integration of the sequence into the chromosomes of the hostcell is desired, it will be possible to use, for example, systems of theplasmid or viral type; such viruses will be, for example, retroviruses(Temin, 1986), or AAVs (Carter, 1993).

Such vectors will be prepared according to the methods commonly used bypersons skilled in the art, and the clones resulting therefrom may beintroduced into an appropriate host by standard methods such as, forexample, lipofection, electroporation or heat shock.

The invention comprises, in addition, the host cells, in particulareukaryotic and prokaryotic cells, transformed by the vectors accordingto the invention, as well as transgenic animals, except humans,comprising one of the said transformed cells according to the invention.

Among the cells which can be used for these purposes, there may ofcourse be mentioned bacterial cells (Olins and Lee, 1993), but alsoyeast cells (Buckholz, 1993), as well as animal cells, in particularmammalian cell cultures (Edwards and Aruffo, 1993), and in particularChinese hamster ovary cells (CHO), but also insect cells in which it ispossible to use methods using baculoviruses, for example (Luckow, 1993).A preferred cellular host for the expression of the proteins of theinvention consists of the CHO cells.

Among the mammals according to the invention, there will be preferredanimals such as mice, rats or rabbits, expressing a polypeptideaccording to the invention, the phenotype corresponding to the normal orvariant LSR receptor, in particular mutated of human origin.

Among the animal models more particularly of interest here, there are inparticular

-   -   transgenic animals exhibiting a deficiency in one of the        components of LSR. They are obtained by homologous recombination        on embryonic stem cells, transfer of these stem cells to        embryos, selection of the chimeras affected at the level of the        reproductive lines, and growth of the said chimeras;    -   transgenic mice overexpressing one or more of the genes for the        LSR complex of murine and/or human origin. The mice are obtained        by transfection of multiple copies of the genes for the LSR        complex under the control of a strong promoter of an ubiquitous        nature, or selective for a type of tissue, preferably the liver;    -   transgenic animals (preferably mice) made deficient in one or        more of the genes for the LSR complex, by inactivation with the        aid of the LOXP/CRE recombinase system (Rohlmann et al., 1996)        or any other system for inactivating the expression of a gene at        a precise age of the animal;    -   animals (preferably rats, rabbits, mice) overexpressing one or        more of the genes for the LSR complex, after viral transcription        or gene therapy;    -   crossings of animals deficient in LSR (in particular mice) with        animals deficient in, or overexpressing:        -   the LDL receptor (Herz et al., 1995; Ishibashi et al.,            1993)>        -   hepatic lipase (Homanics et al., 1995; Kobayashi et al.,            1996)>        -   apoprotein B (Purcellhuynh et al., 1995; Fan et al., 1995)>        -   apoprotein E (Plump et al., 1992; Zhang et al., 1992; Huang            et al., 1996)>        -   apoCIII (Aalto-Setälä et al., 1992; Ito et al., 1990; Maeda            et al., 1994).

The production of transgenic animals, and the viral or nonviraltransfections will be preferably carried out on the following rat andmouse lines:

-   -   Zucker rat (fa/fa) (lida et al., 1996)    -   AKR/J mouse (West et al., 1992)    -   ob/ob mouse (Zhang et al., 1994)    -   ob²j/ob²j mouse (ibid)    -   tubby mouse (Kleyn et al., 1996; Nobben-Trauth et al., 1996)    -   fat/fat (Heldin et al., 1995)    -   agouti mouse (Lu et al., 1994; Manne et al., 1995)    -   db/db mouse (Chen et al., 1996).

The cells and mammals according to the invention can be used in a methodfor the production of a polypeptide according to the invention, asdescribed below, and can also serve as a model for analysis andscreening.

The transformed cells or mammals as described above can also be used asmodels so as to study the interactions between the polypeptides of theLSR complex, between these and their partners, chemical or proteincompounds, which are involved directly or indirectly in the activitiesof the receptor for lipoproteins or the receptor for cytokines, and inparticular for leptin, and in order to study the different mechanismsand interactions called into play according to the type of activity, oraccording to whether a normal complex is involved, or a complex in whichat least one of the domains is a variant.

In particular, they may be used for the selection of products whichinteract with the LSR complex, or one of its normal or variant domains,as cofactor or as inhibitor, in particular a competitive inhibitor, oralternatively having an agonist or antagonist activity on theconformational changes in the LSR complex. Preferably, the saidtransformed cells will be used as a model allowing, in particular, theselection of products which make it possible to combat obesity or thepathologies mentioned above. The said cells may also serve for thedetection of the potential risks posed by certain compounds.

Production of Polypeptides Derived from the LSR Receptor

The invention also relates to the synthesis of synthetic or recombinantpolypeptides of the invention, in particular by chemical synthesis or bythe use of a nucleic acid sequence according to the invention.

The polypeptides according to the present invention can be obtained bychemical synthesis using any of the numerous known peptide syntheses,for example the techniques using solid phases or techniques usingpartial solid phases, by condensation of fragments or by a conventionalsynthesis in solution.

When the compounds according to the present invention are synthesized bythe solid phase method, the C-terminal amino acid is bound to an inertsolid support and comprises groups protecting its amino group at thealpha position (and if necessary, protection on its functional sidegroups).

At the end of this step, the group protecting the amino-terminal groupis removed and the second amino acid, it too comprising the necessaryprotection, is bound.

The N-terminal protecting groups are removed after each amino acid hasbeen bound; on the other hand, the protection is of course maintained onthe side chains. When the polypeptide chain is complete, the peptide iscleaved from its support and the side protecting groups are removed.

The solid phase synthesis technique is well known to a person skilled inthe art. See in particular Stewart et al. (1984) and Bodansky (1984).

The polypeptides obtained by chemical synthesis and which may comprisecorresponding non-natural amino acids are also included in theinvention.

The method for the production of a polypeptide of the invention inrecombinant form is itself included in the present invention, and ischaracterized in that the transformed cells, in particular the cells ormammals of the present invention, are cultured under conditions allowingthe expression of a recombinant polypeptide encoded by a nucleic acidsequence according to the invention, and in that the said recombinantpolypeptide is recovered.

Also forming part of the invention is a method for the production of aheterologous polypeptide, characterized in that it uses a vector or ahost cell containing at least one of the promoter and/or regulatorysequences according to the invention, or at least one of the sequencesfor cellular addressing according to the invention, or one of theirfragments.

The recombinant polypeptides, characterized in that they are capable ofbeing obtained by the said method of production, also form part of theinvention.

The recombinant polypeptides obtained as indicated above may be both inglycosylated and nonglycosylated form and may or may not have thenatural tertiary structure.

These polypeptides may be produced from the nucleic acid sequencesdefined above, according to techniques for the production of recombinantpolypeptides known to persons skilled in the art. In this case, thenucleic acid sequence used is placed under the control of signalsallowing its expression in a cellular host.

An effective system of production of a recombinant polypeptide requireshaving a vector and a host cell according to the invention.

These cells may be obtained by introducing into the host cells anucleotide sequence inserted into a vector as defined above, and thenculturing the said cells under conditions allowing the replicationand/or expression of the transfected nucleotide sequence.

The methods for the purification of a recombinant polypeptide which areused are known to persons skilled in the art. The recombinantpolypeptide may be purified from cell lysates and extracts, from theculture medium supernatant, by methods used individually or incombination, such as fractionation, chromatographic methods,immunoaffinity techniques with the aid of specific mono- or polyclonalantibodies, and the like.

A preferred variant consists in producing a recombinant polypeptidefused with a “carrier” protein (chimeric protein). The advantage of thissystem is that it allows a stabilization and a reduction in proteolysisof the recombinant product, an increase in solubility during in vitrorenaturation and/or simplification of the purification when the fusionpartner has affinity for a specific ligand.

Antibodies

The mono- or polyclonal antibodies or fragments thereof, chimeric orimmunoconjugated antibodies, characterized in that they are capable ofspecifically recognizing a polypeptide or receptor according to theinvention, also form part of the invention.

Specific polyclonal antibodies may be obtained from a serum of an animalimmunized against, for example:

-   -   the LSR receptor purified from membranes of cells carrying the        said LSR receptor, by methods well known to persons skilled in        the art such as affinity chromatography using, for example,        recombinant leptin as specific ligand, or    -   a polypeptide according to the invention, in particular produced        by genetic recombination or by peptide synthesis, according to        the customary procedures, from a nucleic acid sequence according        to the invention.

There may be noted in particular the advantage of antibodiesspecifically recognizing certain polypeptides, variants or fragments,which are in particular biologically active, according to the invention.

The specific monoclonal antibodies may be obtained according to theconventional hybridoma culture method described by Kohler and Milstein,1975.

The antibodies according to the invention are, for example, chimericantibodies, humanized antibodies, Fab or F(ab′)2 fragments. They mayalso be in the form of immunoconjugates or of labelled antibodies so asto obtain a detectable and/or quantifiable signal.

The invention also relates to methods for the detection and/orpurification of a polypeptide according to the invention, characterizedin that they use an antibody according to the invention.

The invention comprises, in addition, purified polypeptides,characterized in that they are obtained by a method according to theinvention.

Moreover, in addition to their use for the purification of polypeptides,the antibodies of the invention, in particular the monoclonalantibodies, may also be used for the detection of these polypeptides ina biological sample.

They thus constitute a means for the immunocytochemical orimmunohistochemical analysis of the expression of the polypeptide of theLSR receptor on specific tissue sections, for example byimmunofluorescence, gold labelling, enzymatic immunoconjugates.

They make it possible in particular to detect abnormal expression ofthese polypeptides in the biological tissues or samples, which makesthem useful for the detection of abnormal expression of the LSR receptoror for monitoring the progress of the method of prevention or treatment.

More generally, the antibodies of the invention may be advantageouslyused in any situation where the expression of a polypeptide of the LSRreceptor, normal or mutated, needs to be observed.

Detection of Allelic Variability and Diagnosis

Also forming part of the invention are the methods for the determinationof an allelic variability, a mutation, a deletion, a loss ofheterozygosity or a genetic abnormality, characterized in that they usea nucleic acid sequence or an antibody according to the invention.

These methods relate to, for example, the methods for the diagnosis ofpredisposition to obesity, to the associated risks, or to pathologiesassociated with abnormalities in the metabolism of cytokines, bydetermining, in a biological sample from the patient, the presence ofmutations in at least one of the sequences described above. The nucleicacid sequences analysed may be either the genomic DNA, the cDNA or themRNA.

It will also be possible to use nucleic acids or antibodies based on thepresent invention in order to allow a positive and differentialdiagnosis in a patient taken in isolation. The nucleic sequences will bepreferably used for a pre-symptomatic diagnosis in an at risk subject,in particular with a familial history. It is also possible to envisagean ante-natal diagnosis.

In addition, the detection of a specific mutation may allow an evolutivediagnosis, in particular as regards the intensity of the pathology orthe probable period of its appearance.

The methods allowing the detection of a mutation in a gene compared withthe natural gene are, of course, highly numerous. They can essentiallybe divided into two large categories The first type of method is that inwhich the presence of a mutation is detected by comparing the mutatedsequence with the corresponding nonmutated natural sequence, and thesecond type is that in which the presence of the mutation is detectedindirectly, for example by evidence of the mismatches due to thepresence of the mutation.

These methods can use the probes and primers of the present inventionwhich are described. They are generally purified nucleic sequences forhybridization comprising at least 8 nucleotides, characterized in thatthey can hybridize specifically with a nucleic sequence chosen from thegroup comprising SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQ ID 7, SEQ ID 9, SEQID 11, SEQ ID 13, SEQ ID 14 SEQ ID 15, SEQ ID 19 and SEQ ID 41.Preferably, the specific hybridization conditions are like those definedin the examples, or such that they ensure at least 95% homology. Thelength of these nucleic sequences for hybridization can vary from 8, 10,15, 20 or 30 to 200 nucleotides, particularly from 20 to 50 nucleotides,more particularly from 20 to 30 nucleotides.

Among the methods for the determination of an allelic variability, amutation, a deletion, a loss of heterozygocity or a genetic abnormality,the methods comprising at least one stage for the so-called PCR(polymerase chain reaction) or PCR-like amplification of the targetsequence according to the invention likely to exhibit an abnormalitywith the aid of a pair of primers of nucleotide sequences according tothe invention are preferred. The amplified products may be treated withthe aid of an appropriate restriction enzyme before carrying out thedetection or assay of the targeted product.

PCR-like will be understood to mean all methods using direct or indirectreproductions of nucleic acid sequences, or alternatively in which thelabelling systems have been amplified, these techniques are of courseknown, in general they involve the amplification of DNA by a polymerase;when the original sample is an RNA, it is advisable to carry out areverse transcription beforehand. There are currently a great number ofmethods allowing this amplification, for example the so-called NASBA“Nucleic Acid Sequence Based Amplification” (Compton 1991), TAS“Transcription based Amplification System” (Guatelli et al., 1990), LCR“Ligase Chain Reaction” (Landegren et al., 1988), “Endo RunAmplification” (ERA), “Cycling Probe Reaction” (CPR), and SDA “StrandDisplacement Amplification” (Walker et al., 1992), methods well known topersons skilled in the art.

The invention comprises, in addition, methods for the diagnosis ofpathologies and/or pathogeneses correlated with abnormal expression of apolypeptide and/or a receptor according to the invention, characterizedin that an antibody according to the invention is brought into contactwith the biological material to be tested, under conditions allowing thepossible formation of specific immunological complexes between the saidpolypeptide and the said antibody, and in that the immunologicalcomplexes possibly formed are detected.

Mutations in one or more genes of the LSR complex may be responsible forvarious modifications of their product(s), which modifications can beused for a diagnostic approach. Indeed, modifications of antigenicitycan allow the development of specific antibodies. The discriminationbetween the various conformations of LSR can be achieved by thesemethods. All these modifications may be used in a diagnostic approach byvirtue of several well-known methods based on the use of mono- orpolyclonal antibodies recognizing the normal polypeptide or mutatedvariants, such as for example using RIA or ELISA.

These diagnostic methods also relate to the methods of diagnosis byimaging in vivo or ex vivo using the monoclonal or polyclonal antibodiesaccording to the invention, particularly those labelled andcorresponding to all or part of the mutated polypeptides (imaging withthe aid of antibodies coupled to a molecule which is detectable inPET-scan type imaging, for example).

Screening of Compounds of Interest

Also included in the invention are the methods for selecting thechemical or biochemical compound capable of interacting, directly orindirectly, with the receptor according to the invention, and/orallowing the expression or the activity of the said receptor to bemodulated, characterized in that they use a receptor, a nucleic acid, apolypeptide, a vector, a cell or a mammal according to the invention.

Screening of Compounds Modifying the Activity of the LSR Receptor

The invention relates to a method for screening compounds modifying theactivity of the LSR receptor, consisting in measuring the effect ofcandidate compounds on various parameters reflecting, directly orindirectly, taken independently or in combination, an LSR receptoractivity.

For the screening of compounds capable of modulating the LSR activityfor lipoprotein clearance, the preferred principal effect is the effectof the compound on the activity of binding, internalization anddegradation of the lipoproteins by the LSR receptor.

This effect can be analysed in the absence or in the presence of freefatty acids, or of any other agent known to induce or to inhibit theactivity of LSR on the clearance of lipoproteins, or in the absence orthe presence of leptin, or of any other agent capable of inducing or ofinhibiting the LSR function of cytokine clearance. It can, in addition,be measured in the absence or in the presence of agents capable ofpromoting or reducing the lipase activities, either intracellular orextracellular, as well as in the presence or in the absence ofalternative known routes of degradation of lipoproteins.

Various indirect parameters can also be measured, including thefollowing

-   -   the change in weight induced by the administration of the        compound    -   the food intake induced by the administration of the compound    -   the postprandial lipemic response induced by the administration        of the compound, before, during or after ingestion of a meal,        for example high in fat.

The selection of compounds capable of influencing the plasmatriglyceride concentrations, and/or the binding, internalization andhepatic degradation of lipoproteins or particles high in triglycerides,will be preferred.

For the screening of compounds capable of modulating the LSR activity ofclearance of cytokines, in particular of leptin, the preferred principaleffect is the effect of the compound on the activity of binding,internalization and hepatic degradation of cytokines by the LSRreceptor, in the absence or in the presence of free fatty acids.

The measurement of the binding, internalization and/or degradation oflipids or of cytokines can be carried out, for example, on hepatocytesor fibroblasts in culture, or on any other cell expressing the LSRreceptor at its surface. The cells will be preferably cells expressing arecombinant LSR receptor, more particularly cells expressing arecombinant LSR receptor and whose endogenous LSR receptor would beinactivated or absent. These cells may or may not express the LDLreceptor.

The screening of compounds modulating the LSR activity preferably usescells or model animals according to the invention, in particular mice,rats or humans, more particularly those described above and in theexamples which follow.

Screening of Compounds Modifying the Expression of the LSR Receptor

Screening may be used to test compounds capable of modifying the leveland/or the specificity of expression of the LSR receptor either bybinding competitively to the sites for binding of transcription factorssituated in the LSR promoter or by binding directly to the transcriptionfactors.

The level of expression of the LSR receptor and its location can beanalysed by hybridization in solution with large probes as indicated inPatent PCT WO 97/05277, the teaching of this document being incorporatedby reference. Briefly, a cDNA or the genomic DNA for the LSR receptor oralternatively a fragment thereof is inserted at a cloning site situateddirectly downstream of a bacteriophage (T3, T7 or SP6) RNA polymerasepromoter in order to produce an antisense RNA. Preferably, the insertcomprises at least 100 consecutive nucleotides of the genomic sequenceof the LSR receptor or of one of the cDNAs of the present invention,more particularly one or more of the cDNAs of SEQ ID 9, SEQ ID 11 or SEQID 13. The plasmid is linearized and transcribed in the presence ofribonucleotides comprising modified ribonucleotides such as Biotin-UTPand Digoxigenin-UTP. An excess of this labelled RNA is hybridized insolution with the mRNAs isolated from cells or from tissues of interest.The hybridizations are carried out under stringent conditions (40-50° C.for 16 h in a solution containing 80% formamide and 0.4 M NaCl, pH 7-8).The non-hybridized probe is eliminated by digestion with ribonucleasesspecific for single-stranded RNAs (CL3, T1, PhyM, U2 or A RNases). Thepresence of modified nucleotides biotin-UTP allows the capture of thehybrids on microtitre plates carrying streptavidine. The presence of theDIG modification allows the detection and quantification of the hybridsby ELISA using anti-DIG antibodies coupled to alkaline phosphatase.

A quantitative analysis of the expression of the gene for the LSRreceptor can also be carried out using DNA templates, the term DNAtemplates designating a one-dimensional, two-dimensional ormulti-dimensional arrangement of a plurality of nucleic acids having asufficient length to allow a specific detection of the expression ofmRNAs capable of hybridizing thereto. For example, the DNA templates maycontain a plurality of nucleic acids derived from genes for which it isdesired to estimate the level of expression. The DNA templates mayinclude the genomic sequences of LSR, that of a cDNA of the presentinvention, more particuliarly one or more of the cDNAs of SEQ ID 9, SEQID 11 or SEQ ID 13, any sequences complementary thereto or any fragmentsthereof. Preferably, the fragments comprise at least 15, at least 25, atleast 50, at least 100 or at least 500 consecutive nucleotides of thenucleic sequences from which they are derived.

For example, a quantitative analysis of the expression of the LSRreceptor can be carried out with a DNA template having the cDNA for theLSR receptor as described in Schena et al. (1995 and 1996). cDNAs forthe LSR receptor or fragments thereof are amplified by PCR and bound inthe form of a template from a 96-well microplate onto a sylatedmicroscope slide using a very fast automated machine. The DNA templatethus produced is incubated in a humid chamber in order to allow itsrehydratation. It is then rinsed once in 0.2% SDS for 1 min, twice inwater for 1 min and once for 5 min in a sodium borohydride solution. Thetemplate is then submerged in water for 2 min at 95° C., transferredinto 0.2% SDS for 1 min, rinsed twice with water, dried and stored inthe dark at 25° C.

The mRNAs of cells and of tissues are isolated or obtained from acommercial source, for example the company Clontech. The probes areprepared by a reverse transcription cycle. The probes are thenhybridized with the DNA template of 1 cm² under a glass coverslip of14×14 mm for 6-12 hours at 60° C. The template is washed for 5 min at25° C. in a washing buffer at low stringency (1×SSC/0.2% SDS) and thenfor 10 min at room temperature in a highly stringent buffer(0.1×SSC/0.2% SDS). The template is analysed in 0.1×SSC using a laserfluorescence microscope with a set of appropriate filters. Measurementsof precise differential expression are obtained by taking the mean ofthe ratios of two independent hybridizations.

A quantitative analysis of the expression of the LSR receptor can alsobe carried out with cDNAs for the LSR receptor or fragments thereof onDNA templates according to the description by Pietu et al. (1996). ThecDNAs for the LSR receptor or fragments thereof are amplified by PCR andbound to membranes. The mRNAs obtained from different tissues or cellsare labelled with radioactive nucleotides. After hybridization andwashing under controlled conditions, the hybridized mRNAs are detectedwith a Phosphor Imager or by autoradiography. The experiments arecarried out in duplicate and a quantitative analysis of thedifferentially expressed mRNAs can be carried out.

Alternatively, the analysis of the expression of the LSR receptor can bemade with DNA templates at high density as described by Lockhart et al.(1996) and Sosnowski et al. (1997). Oligonucleotides of 15 to 50nucleotides, preferably about 20 nucleotides, extracted from genomic DNAor cDNA sequences for the LSR receptor or of their complementarysequences are synthesized directly on a chip or synthesized and thenaddressed onto the chip.

LSR cDNA probes labelled with an appropriate compound such as biotin,digoxigenin or a fluorescent molecule are synthesized from a populationof mRNA and are fragmented into oligonucleotides of 50 to 100nucleotides on average. The probes thus obtained are then hybridized toa chip. After washing as described in Lockhart et al (1996) and anapplication of various electric fields (Sosnowski et al. 1997), thelabelled compounds are detected and quantified. The hybridizations areduplicated. A comparative analysis of the intensity of the signalsgenerated by the probes on the same target oligonucleotide in variouscDNA samples indicates a differential expression of the mRNAs for theLSR receptor.

The techniques mentioned above allow the analysis of the levels ofexpression of the LSR receptor, in the same cell or the same tissuedepending on various conditions, for example of induction or ofnoninduction, but also the analysis of the tissue specificity of thisexpression, under conditions which can also vary. It will be possible,by virtue of these techniques, to analyse the expression of either ofthe subunits of the LSR receptor, and more generally of different formsderived from alternative splicing, by adequately defining the probes.

The effect of compounds which are candidates for modulating the level orthe specificity of expression, or of splicing of the different forms ofthe LSR receptor can thus be analysed on a large scale by exposing thecells which are the source of messenger RNA, in particular the modelcells according to the invention, whether they express LSR naturally orwhether they are recombinant cells, to the said candidate compounds.

Screening of Compounds Interacting With the LSR Receptor

Another aspect of the present invention consists in methods ofidentifying molecules capable of binding to the LSR receptor. Suchmolecules can be used to modulate the activity of the LSR receptor. Forexample, such molecules can be used to stimulate or reduce thedegradation of lipoproteins, preferably of lipoproteins high intriglycerides, or of cytokines, preferably of leptin. Such molecules canalso be used to inhibit the activation by leptin or the activation byfree fatty acids of the LSR activity.

Numerous methods exist for identifying ligands for the LSR receptor. Oneof these methods is described in patent U.S. Pat. No. 5,270,170, whoseteaching is incorporated by reference. Briefly, a library is constructedwhich consists of random peptides, comprising a plurality of vectorseach encoding a fusion between a peptide which is a candidate forbinding to the LSR receptor and a protein binding to DNA such as the Lacrepressor encoded by the lad gene. The vectors for the library of randompeptides also contain binding sites for the proteins binding to DNA suchas the LacO site when the protein is the Lac repressor. The library ofrandom peptides is introduced into a host cell in which the fusionprotein is expressed. The host cell is then lysed under conditionsallowing the binding of the fusion protein to the sites of the vector.

The vectors which have bound the fusion protein are brought into contactwith the immobilized LSR receptor, a subunit of the immobilized LSRreceptor or a fragment of the immobilized LSR receptor under conditionsallowing the peptides to bind specifically. For example, the LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acidscan be immobilized by binding to a surface such as a plate or a plasticparticle.

The vectors which encode the peptides capable of binding to the LSRreceptor are specifically retained at the surface by interactionsbetween the peptide and the LSR receptor, a subunit of the receptor or afragment thereof.

Alternatively, molecules capable of interacting with the LSR receptorcan be identified using a double hybrid system such as the MatchmakerTwo Hybrid System 2. According to the instructions of the manualaccompanying the Matchmaker Two Hybrid System 2 (Catalogue No. K1604-1,Clontech), whose teaching is incorporated by reference, the nucleicacids encoding the LSR receptor, a subunit thereof or a fragment thereofcomprising at least 10, at least 20, at least 30, or more than 30consecutive amino acids are inserted into an expression vector so thatthey are in phase with the DNA encoding the DNA binding domain of thetranscription activator of yeast GAL4. The nucleic acids of a libraryencoding proteins or peptides capable of interacting with the LSRreceptor are inserted into a second expression vector so that they arein phase with the DNA encoding the activation domain of the GAL4activator. The yeasts are transformed with the two expression plasmidsand they are placed in a medium which makes it possible to select thecells expressing markers contained in each of the vectors as well asthose expressing the HIS3 gene whose expression is dependent on GAL4.The transformed cells capable of growing on a histidine-free medium areanalysed for expression of LacZ under the dependence of GAL4. The cellswhich grow in the absence of histidine and express LacZ contain aplasmid which encodes proteins or peptides which interact with the LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acidsthereof.

To study the interaction of the LSR receptor, a subunit thereof or afragment thereof comprising at least 10, at least 20, at least 30, ormore than 30 consecutive amino acids with small molecules such as thosegenerated by combinatory chemistry, it is possible to use anHPLC-coupled microdialysis as described in Wang et al. (1997), or anaffinity capillary electrophoresis as described in Busch et al. (1997),the teaching of these documents being incorporated by reference.

In other methods, the peptides or small molecules capable of interactingwith the LSR receptor, a subunit thereof or a fragment thereofcomprising at least 10, at least 20, at least 30, or more than 30consecutive amino acids may be linked to detectable markers such asradioactive, fluorescent or enzymatic markers. These labelled moleculesare brought into contact with the immobilized LSR receptor, animmobilized subunit thereof or an immobilized fragment thereofcomprising at least 10, at least 20, at least 30, or more than 30consecutive amino acids under conditions allowing a specificinteraction. After elimination of the molecules which are notspecifically bound, the bound molecules are detected by appropriatemeans.

These methods may allow in particular the identification of fatty acidsor analogues capable of binding to the fatty acid binding site on theLSR, of lipoproteins or analogues, capable of binding to the lipoproteinbinding site on the LSR receptor, of leptin derivatives or analoguescapable of binding to the leptin binding site on the LSR, and ofderivatives of the gC1qR receptor or analogues capable of binding to thegC1qR binding site on the LSR.

In addition, the peptides or small molecules which bind to LSR,preferably to the binding sites on the LSR receptor for fatty acids,lipoproteins, cytokines, in particular leptin, or gC1qR or one of itsanalogous proteins, can be identified by competition experiments. Insuch experiments, the LSR receptor, a subunit thereof or a fragmentthereof comprising at least 10, at least 20, at least 30, or more than30 consecutive amino acids is immobilized on a surface such as a plasticsupport. Increasing quantities of peptides or of small molecules arebrought into contact with the immobilized LSR receptor, an immobilizedsubunit thereof or an immobilized fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acids inthe presence of a labelled ligand for the receptor, it being possiblefor this ligand to be, for example, leptin, oleate, the LDLs or gC1qR.The ligand for the LSR receptor may be labelled with a radioactive,fluorescent or enzymatic marker. The capacity of the molecule tested tointeract with the LSR receptor, a subunit thereof or a fragment thereofcomprising at least 10, at least 20, at least 30, or more than 30consecutive amino acids is determined by measuring the quantity oflabelled ligand bound in the presence of the molecule tested. A decreasein the quantity of bound ligand when the molecule tested is presentindicates that the latter is capable of interacting with the LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acids.

These methods can in particular allow the identification of fatty acidsor analogues capable of binding to the fatty acid binding site on theLSR, of lipoproteins or analogues, capable of binding to the lipoproteinbinding site on the LSR receptor, of leptin derivatives or analoguescapable of binding to the leptin binding site on the LSR, and ofderivatives of the gC1qR receptor or analogues capable of binding to thegC1qR binding site on the LSR. The capacity of such compounds, or of anyother candidate compound, to compete with the binding of oleates,lipoproteins, leptin or gC1qR to LSR can be measured in particular.

The BIACORE technology can also be used to carry out the screening ofcompounds capable of interacting with the LSR receptor. This technologyis described in Szabo et al. (1995) and in Edwards and Leartherbarrow(1997), of which the teaching is incorporated by reference, and makes itpossible to detect interactions between molecules in real time withoutthe use of labelling. It is based on the phenomenon of SPR (surfaceplasmon resonance). Briefly, the molecule to be analysed is bound to asurface (typically using a carboxymethyl dextran matrix). A light ray isdirected onto the face of the surface which does not contain the sampleand is reflected by the said surface. The SPR phenomenon causes areduction in the intensity of the reflected light with a specificcombination of angle and of wavelength. The molecule binding eventscause a change in the refractive index at the surface which is detectedas a modification of the SPR signal. To carry out a screening ofcompounds capable of interacting with the LSR receptor, the LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acids,is immobilized on a surface. This surface constitutes one face of a cellthrough which passes the molecule to be tested. The binding of themolecule to the LSR receptor, a subunit thereof or a fragment thereofcomprising at least 10, at least 20, at least 30, or more than 30consecutive amino acids is detected by a change in the SPR signal. Themolecules tested may be proteins, peptides, carbohydrates, lipids orsmall molecules generated, for example, by combinatory chemistry. Thecandidate proteins can be extracted from any tissue, obtained from anyspecies. The BIACORE technology can also be used by immobilizingeukaryotic or prokaryotic cells or lipid vesicles having an endogenousor recombinant LSR receptor at their surface.

One of the main advantages of this method is that it allows thedetermination of the association constants between the LSR receptor andthe interacting molecules. Thus, it is possible to specifically selectthe molecules interacting with high or low association constants.

The proteins or other molecules interacting with the LSR receptor, asubunit thereof or a fragment thereof comprising at least 10, at least20, at least 30, or more than 30 consecutive amino acids can beidentified using affinity columns which contain the LSR receptor, asubunit thereof or a fragment thereof comprising at least 10, at least20, at least 30, or more than 30 consecutive amino acids. The LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acidsmay be attached to the column using conventional techniques includingchemical coupling to an appropriate column matrix such as agarose, AffiGel, or other matrices known to a person skilled in the art. In anotheraspect of the invention, the affinity column may contain chimericproteins in which the LSR receptor, a subunit thereof or a fragmentthereof comprising at least 10, at least 20, at least 30, or more than30 consecutive amino acids would be fused, for example, with glutathioneS-transferase. The molecules to be tested which are described above arethen deposited on the column. The molecules interacting with the LSRreceptor, a subunit thereof or a fragment thereof comprising at least10, at least 20, at least 30, or more than 30 consecutive amino acidsare retained by the column and can be isolated by elution. In the casewhere the molecules tested are proteins, they can then be analysed on a2-D electrophoresis gel as described in Ramunsen et al. (1997), of whichthe teaching is incorporated by reference. Alternatively, the proteinsor the other molecules retained by the affinity column can be purifiedby electrophoresis and sequenced. A similar method can be used toisolate antibodies, to screen “phage display”, products or “phagedisplay” derived human antibodies.

Screening of Compounds Interacting with the Promoter and/or RegulatorySequences of the LSR Receptor

The invention also relates to a method of screening compoundsinteracting with the promoter and/or regulatory sequences of the LSRreceptor.

The nucleic acids encoding proteins interacting with the promoter and/orregulatory sequences of the LSR receptor gene, more particularly anucleotide sequence corresponding to nucleotides 1 to 1897 of SEQ ID 19or a fragment thereof, can be identified using a single hybrid systemsuch as that described in the manual accompanying the MatchmakerOne-Hybrid System from Clontech (Catalogue No. K1603-1), of which theteaching is incorporated by reference. Briefly, the target nucleotidesequence is cloned upstream of a selectable marker gene and integratedinto a yeast genome. The yeasts containing the integrated marker geneare transformed by a library containing fusions between cDNAs encodingcandidate proteins for binding to the promoter and/or regulatory regionsof the gene for the LSR receptor and the yeast transcription factoractivating domain such as GAL4. The yeasts are placed in a medium whichmakes it possible to select the cells expressing the marker gene. Theyeasts selected contain a fusion protein capable of binding to thepromoter and/or regulatory target region. The cDNAs of the genesencoding the fusion proteins are then sequenced. The correspondinginserts can then be cloned into expression or transcription vectors invitro. The binding of the polypeptides thus encoded to the promotertarget sequences can be confirmed by techniques familiar to personsskilled in the art, including gel retardation or protection to DNAseexperiments.

The screening of compounds capable of modifying the expression of theLSR receptor by binding to its regulatory and/or promoter sequences canalso be carried out with the aid of “reporter” genes. For example, agenomic region situated in 5′ of the coding sequence of the LSRreceptor, more particularly a nucleotide sequence corresponding tonucleotides 1 to 1897 of SEQ ID 19 or a fragment thereof, can be clonedinto a vector such as pSEAP-Basic, pSEAP-Enhancer, pβgal-Basic,pβgal-Enhancer, or pEGFP-1 available from Clontech. Briefly, each ofthese vectors contains multiple cloning sites situated upstream of amarker gene encoding an easily detectable protein such as alkalinephosphatase, β-galactosidase or GFP (green fluorescent protein). Afterinsertion of the genomic region situated in 5′ of the coding sequence ofthe LSR receptor, more particularly a nucleotide sequence correspondingto nucleotides 1 to 1897 of SEQ ID 19 or a fragment thereof, the levelof expression of the marker proteins is measured and compared with avector containing no insert. The effect of candidate compounds on theexpression resulting from the regulatory and/or promoter sequences ofLSR can thus be evaluated.

The screening of the compounds capable of binding to the regulatoryand/or promoter regions of the gene for the LSR receptor can also becarried out by gel retardation experiments well known to persons skilledin the art and described in Fried and Crothers (1981), Garner and Revzin(1981) and Dent and Latchman (1993), of which the teaching isincorporated by reference. These experiments are based on the principlethat a DNA fragment bound to a protein migrates more slowly than thesame fragment without protein. Briefly, the target nucleotide sequenceis labelled. It is then brought into contact either with a nuclear ortotal cell extract prepared so as to contain the transcription factors,or with various compounds to be tested. The interaction between theregulatory and/or promoter region of the gene for the LSR receptor andthe transcription factor or compound is detected after electrophoresisby retardation of migration.

Compounds

The chemical or biochemical compounds, characterized in that they makeit possible to modulate the expression or the activity of the receptoraccording to the invention, also form part of the invention.

The chemical or biochemical compounds, characterized in that they arecapable of interacting, directly or indirectly, with the receptoraccording to the invention, also form part of the invention.

The chemical or biochemical compounds, characterized in that they areselected by the said methods defined above, also form part of theinvention.

In particular, among these compounds according to the invention, aleptin or one of its derived compounds, preferably one of its proteinvariants, or leptins which are chemically modified or which are obtainedby genetic recombination, or one of their fragments, are preferred.

Compounds which make it possible to modulate the expression or theactivity of the receptor are understood to mean the compounds which makeit possible in particular to reduce, stabilize or increase the number,the recycling rate and/or the change in the conformation of the receptoraccording to the invention, or to promote or inhibit the overallactivity or the activity of one of the domains of the said receptor oralternatively to reestablish normal expression of the said receptor inthe case, for example, where a genetic abnormality is observed. Thesecompounds may, for example, interact as ligands specific for the saidreceptor or for one of its domains as cofactor, or as inhibitor, inparticular a competitive inhibitor, or alternatively having an agonistor antagonist activity on the conformational changes in the complex.These compounds may also interact by neutralizing the natural ligandsspecific for the said receptor and by thereby inhibiting the receptoractivity induced by these ligands.

Among these compounds, the compounds which make it possible to modulatethe number of polypeptides of the said receptor, its recycling rateand/or the selectivity of their activity, are preferred.

Also preferred are the compounds according to the invention,characterized in that they allow an increase in the total activity or inthe expression of the receptor according to the invention, and/or aspecific increase in the clearance activity for cytokines, in particularleptin, of the said receptor, and/or a specific increase in theclearance activity for lipoproteins, of the said receptor.

Also preferred are the compounds characterized in that they allow adecrease in the total activity or in the expression of the receptoraccording to the invention, and/or a specific decrease in the clearanceactivity for cytokines, in particular leptin, of the said receptor,and/or a specific decrease in the clearance activity for lipoproteins,of the said receptor.

Also preferred are the compounds characterized in that they allowmodulation of the elimination of the cytokines, in particular leptin,and/or modulation of the elimination of the lipoproteins, chylomicronresidues, and/or triglycerides.

The invention also comprises the compounds according to the invention,characterized in that they allow modulation of the level of cytokines,in particular leptinemia, and/or modulation of the level oflipoproteins, chylomicron residues, and/or triglycerides.

The compounds according to the invention, characterized in that theyallow control of the level of cytokines, in particular leptinemia, aremore particularly preferred.

Still preferably, the invention comprises the compounds according to theinvention, characterized in that they allow control, preferably adecrease, of the level of lipoproteins, a decrease in the plasmaconcentration of chylomicron residues, and/or a decrease intriglyceridernia.

Among the compounds which are most preferred, there are preferred thosecharacterized in that they are chosen from:

-   a. an antibody according to the invention;-   b. a polypeptide according to the invention;-   c. a polypeptide according to the invention, characterized in that    it corresponds to a soluble form of the receptor according to the    invention;-   d. a vector according to the invention;-   e. a vector according to the invention, characterized in that it has    on its outer surface a site for specific recognition of hepatic    cells;-   f. a vector according to the invention, characterized in that the    product of expression of the nucleic acid inserted by the vector    into the target cell is either anchored in or excreted by the said    transformed target cell;-   g. a sense or antisense oligonucleotide according to the invention;-   h. a leptin, or one of its protein variants, or a leptin which is    chemically modified or which is modified by genetic recombination,    or one of their fragments.

The invention finally relates to the compounds according to theinvention as a medicament.

The compounds according to the invention as a medicament for theprevention and/or treatment of pathologies and/or of pathogeneses linkedto disorders in dietary habit are preferred in particular.

The compounds according to the invention as a medicament for theprevention and/or treatment of pathologies and/or of pathogeneses linkedto disorders in the metabolism of cytokines are also preferred.

Preferably, the invention also relates to the compounds according to theinvention as medicament for the prevention or treatment of obesity oranorexia.

The compounds according to the invention as a medicament for theprevention and/or treatment of pathologies and/or of pathogenesesassociated with, or induced by obesity, are the preferred compounds.

In particular, there are preferred the compounds according to theinvention, as a medicament for the prevention and/or treatment ofcardiac insufficiency, of coronary insufficiency, of cerebrovascularaccidents, of atheromatous disease, of atherosclerosis, of high bloodpressure, of non-insulin-dependent diabetes, of hyperlipidemia and/or ofhyperuricemia.

The most preferred are the compounds according to the invention, as amedicament for the prevention and/or treatment of atheromatous diseaseand/or of atherosclerosis.

Finally, the invention comprises compounds according to the inventionfor the prevention and/or treatment by gene therapy, of pathologiesand/or of pathogeneses linked to disorders in dietary habit, of obesityand/or of pathologies and/or of pathogeneses associated with, or inducedby, obesity.

The compounds of the invention as active ingredients of a medicamentwill be preferably in soluble form, combined with a pharmaceuticallyacceptable vehicle.

Such compounds which can be used as a medicament offer a new approachfor preventing and/or treating pathologies and/or pathogeneses linked todisorders in dietary habit such as obesity or anorexia, and the relatedrisks and/or complications.

Preferably, these compounds will be administered by the systemic route,in particular by the intravenous route, by the intramuscular orintradermal route or by the oral route.

Their modes of administration, optimum dosages and galenic forms can bedetermined according to the criteria generally taken into account inestablishing a treatment suited to a patient, such as for example theage or body weight of the patient, the seriousness of his generalcondition, the tolerance to treatment and the side effects observed, andthe like.

As mentioned above, depending on the cases, it may be advisable toamplify the activity of LSR, by promoting, for example, the expressionof its genes or by increasing the activity of their expression products,in pathological cases resulting from the fact that at least one of thesegenes is not expressed, is insufficiently expressed or is expressed inan abnormal form which does not allow the expression product to carryout its functions, or on the contrary to repress an overexpression or anabnormal expression of these genes. It is therefore advisable in generalto compensate for the deficiency or the overexpression of expressionproducts of this gene by a so-called “replacement” therapy allowing theamplification or the reduction in the activities of the LSR complex.

The replacement therapy may be carried out by gene therapy, that is tosay by introducing the nucleic acid sequences according to the inventionand/or the corresponding genes with the elements which allow theirexpression in vivo, in the case where one of the genes is insufficientlyexpressed for example, or alternatively when it is expressed in anabnormal form.

The principles of gene therapy are known. It is possible to use viralvectors according to the invention; it is also possible to envisagenonviral, that is to say synthetic, vectors which mimic viral sequencesor alternatively which consist of naked RNA or DNA according to thetechnique developed in particular by the company VICAL.

In most cases, it is necessary to envisage targeting elements ensuringexpression specific for the liver so as to be able to limit the zones ofexpression of the proteins which remain involved in the clearance ofleptin and that of lipoproteins. It is even advantageous, in some cases,to have vectors for transient expression or at least for controlledexpression which it will be possible to block when necessary.

Other characteristics and advantages of the invention appear in theremainder of the description with the examples and figures whose legendsare represented below.

LEGEND TO THE FIGURES

FIG. 1: Schematic representation of the three forms of the rat LSRprotein: LSR 66 (α subunit), LSR 64 (α′ subunit), and LSR 58 (αsubunit).

FIG. 2: Alignment of the protein sequences of the long forms (αsubunits) of the human LSR (LSR1.Hs; SEQ ID NO:8), rat LSR (LSR1.Rn; SEQID NO:2) and mouse LSR (LSR1.Mm; SEQ ID NO:16). The (*) symbols placedunder the alignments indicate the conserved amino acids, the (.) symbolsindicate the conservative substitutions of amino acids. Boxed, from theNH₂-terminal end to the COOH-terminal end, the potential fatty acid(FFA) binding site boxed, the clathrin binding site [NPGY], the lyosomaladdressing consensus: di-leucine LI-X10-LL, the transmembrane TM domainoverlined, the motif [RSRS], the potential lipoprotein binding site(+−+−) boxed. Overlined, the signature of the TNF receptor with (arrow);indicated, the amino acids conserved in the signature. The transmembranedomain is situated between the last di-leucine and the TNF signature. A:Alignment shown from amino acid positions 1 to 539 of SEQ ID NO:8. B:Alignment shown from amino acid positions 540 to 649 of SEQ ID NO:8.

FIG. 3: Alignment of the protein sequences of the three types ofsubunits of the human LSR (α: LSR1.Hs, SEQ ID NO:8; α′: LSR2.Hs, SEQ IDNO:10; β: LSR3.Hs, SEQ ID NO:12). The meaning of the symbols, of theboxes and of the overlines is the same as that in FIGS. 2A and 2B. A:Alignment shown from amino acid positions 1 to 540 of SEQ ID NO:8. B:Alignment shown from amino acid positions 541 to 649 of SEQ ID NO:8.

FIG. 4: Alignment of the protein sequences of the three types ofsubunits of rat LSR. (α: LSR1.Rn, SEQ ID NO:2; α′: LSR2.Rn, SEQ ID NO:4;β: LSR3.Rn, SEQ ID NO:6). The meaning of the symbols, of the boxes andof the overlines is the same as that in FIGS. 2A and 2B. A: Alignmentshown from amino acid positions 1 to 540 of SEQ ID NO:2. B: Alignmentshown from amino acid positions 541 to 593 of SEQ ID NO:2.

FIG. 5: Alignment of the protein sequences of the three types ofsubunits of mouse LSR (α: LSR1.Mm, SEQ ID NO:16; α′: LSR2.Mm, SEQ IDNO:17; β: LSR3.Mm, SEQ ID NO:18). The meaning of the symbols, of theboxes and of the overlines is the same as that in FIGS. 2A and 2B. A:Alignment shown from amino acid positions 1 to 540 of SEQ ID NO:16. B:Alignment shown from amino acid positions 541 to 594 of SEQ ID NO:16.

FIG. 6: Schematic representation of the three LSR forms identified inhumans, indicating the motifs conserved on each of them. A: Schematicrepresentation of the genomic organization of the human LSR startingfrom the first coding exon. The exons are indicated by boxes, theintrons by interrupted bars. The size, in nucleotides, of the exons andintrons is indicated above them. The elements characterizing themessenger and the encoded protein are presented in this figure. The boxon the right gives the meaning of the symbols used. B: Structure of theLSR-Hs-2062 form of human LSR. This form encodes a protein of 649 aminoacids. C: Structure of the LSR-Hs-2005 form of human LSR. This formencodes a protein of 630 amino acids. D: Structure of the LSR-Hs-1858form of human LSR. This form encodes a protein of 581 amino acids.

FIG. 7: Alignment of the nucleotide sequences of the long forms of cDNA(encoding the α subunit) or portions thererof for human LSR (Isr1.HS;nucleotides 1 to 2062 of SEQ ID NO:7), rat LSR (Isr1.Rn; SEQ ID NO:1)and mouse LSR (Isr1.Mm; SEQ ID NO:13). The nucleotides conserved in thethree sequences are identified by an * sign placed under the sequences.Dashes are added inside the sequences when the optimum alignment of thesequences cannot be achieved without creating microdeletions. A:Alignment shown from amino acid positions 1 to 486 of SEQ ID NO:1. B:Alignment shown from amino acid positions 487 to 1026 of SEQ ID NO:1. C:Alignment shown from amino acid positions 1027 to 1551 of SEQ ID NO:1.D: Alignment shown from amino acid positions 1552 to 2080 of SEQ IDNO:1. E: Alignment shown from amino acid positions 2081 to 2097 of SEQID NO:1.

FIG. 8: Identification of the LSR receptor by ligand and Westernblotting on solubilized proteins of rat liver membranes (lanes 1, 2 and4), or on the partially purified protein of 240 kD (lane 3). Lanes 1, 2and 3: Ligand blotting. Lane 1: in the absence of oleate and of¹²⁵I-LDL; lane 2: in the presence of oleate and of ¹²⁵I-LDL; lane 3: inthe presence of oleate and of ¹²⁵I-LDL. Lane 4: Western blotting withanti-LSR antibodies.

FIG. 9: Effect of anti-LSR antibodies on the LSR activity. A. Binding of¹²⁵I-LDL onto the plasma membranes of rat hepatocytes in the presence ofoleate and of increasing concentrations of anti-LSR antibody (ν) or ofcontrol antibody (∘), expressed as % of the total quantity of ¹²⁵I-LDLbound in the absence of antibodies. B. Binding, incorporation anddegradation of ¹²⁵I-LDL in rat hepatocytes in primary culture in thepresence of oleate and of anti-LSR antibody (ν) or of control antibody(∘), expressed respectively as % of the binding, incorporation and totaldegradation of ¹²⁵I-LDL in the presence of non-specific antibodies.

FIG. 10: Identification of the LSR receptor by immunoprecipitation of³⁵S-methionine- and ³⁵S-cysteine-labelled hepatocyte lysates, in thepresence of control antibodies (lane 1), or of anti-LSR antibodies(lanes 2 to 4), after separation by electrophoresis under nonreducing(lanes 2 and 3) or reducing (lanes 1 and 3) conditions.

FIG. 11: Cloning of the cDNA encoding α and β-LSR. A. Northern-blotanalysis showing several sizes of LSR messenger RNA. B. Multi-tissueNorthern-blot analysis of LSR mRNA with a probe specific for LSR and acontrol probe specific for β-actin. C. RT-PCR analysis of LSR mRNA using5 pairs of primers covering the entire sequence and identification ofthree forms derived from alternative splicing in the amplificationfragment obtained by means of the bc′ primers. The diagram representsthe results of sequence analysis of the three corresponding forms of LSRcDNA: the squared region is absent from the two short forms, the hatchedregion is absent only from the shortest form.

FIG. 12: Translation in vitro of the two complete cDNAs encoding thelongest (66 kDa, lane 2) and the shortest (58 kDa, lane 3) forms of ratLSR, and of a control cDNA, an antisense of the cDNA encoding thelongest form of LSR (lane 1). The products of translation in vitro,labelled with ³⁵S-methionine, are analysed after electrophoresis undernonreducing conditions.

FIG. 13: Identification of the α and β-LSR subunits as being responsiblefor the LSR activity. A. Diagram showing the location and the sequenceof LSR N-terminal peptide used to generate anti-LSR peptide antibodies.B. Effect of antibodies directed against a synthetic LSR peptide on theLSR activity of rat liver plasma membranes. The LSR activity is measuredin the presence of a control antibody (∘) or of the anti-LSR peptideantibody (ν). C. Western and Ligand blotting of the α and β subunits ofLSR. The Western blotting is carried out using the anti-LSR (lane 1) oranti-LSR peptide (lane 2) antibody. The ligand blotting is carried outin the presence of ¹²⁵I-LDL, with (lane 4) or without (lane 3) oleate.

FIG. 14: Identification of the subunits of the LSR receptor andinhibitory effect of antibodies directed against a C-terminal syntheticpeptide derived from LSR. A—Diagram showing the location and thesequence of the synthetic peptide 170. B—Western blotting of rathepatocyte lysates using antibodies directed against the syntheticpeptide 170 (lane 2), or a control antibody (lane 1); lane 3: molecularweight markers. C—Binding of ¹²⁵I-LDL by the LSR receptor in thepresence of oleate and of control antibodies or antibodies directedagainst the LSR 170 peptide.

FIG. 15: Effect of a transient transfection of CHO-K1 cells with theplasmids expressing the α and β subunits of the LSR receptor on thebinding of LDLs in the presence or in the absence of oleate. Increasingconcentration of α plasmid alone (◯□) fixed concentration of α plasmidand increasing concentration of β plasmid (●▪).

FIG. 16: Effect of a transient transfection of CHO-K1 cells withplasmids expressing the α and β subunits of the LSR receptor on theinternalization and degradation of LDLs. Increasing concentration of αplasmid alone (▪); fixed concentration of α plasmid and increasingconcentration of β plasmid (●). The results are expressed as thedifference between the measurements in the presence and in the absenceof oleate.

FIG. 17: Characterization of the LSR activity obtained in CHO-K1 cellstransiently transfected with the nucleic sequences encoding the α and βsubunits of the LSR receptor, compared with the LSR activity obtained inthe same cells not transfected (control). A—Binding of ¹²⁵I-LDL in thepresence of a control antibody or of an anti-LSR antibody. B—Binding of¹²⁵I-LDL in the presence of increasing concentrations of unlabelledlipoproteins; rat chylomicrons (υ), human VLDL (▪), LDL (□), HDL (σ),LDLs treated with pronase (◯), or LDLs modified with cyclohexanedione(LDL-chd, ●).

FIG. 18: Effect of oleate, of RAP-39, of anti-LSR antibodies and ofchloroquine on the specific degradation of leptin in primary cultures ofrat hepatocytes.

FIG. 19: Western blot analysis with anti-LSR antibodies, of the fractionof rat liver plasma membrane proteins retained on an affinitychromatography column containing leptin.

FIG. 20: Clearance of ¹²⁵I-leptin on control (∘), ob/ob (ν) and db/db (

) mice in the liver and the kidney. The results are expressed as thedifference between the quantities of ¹²⁵I-leptin and¹²⁵I-β2-microglobulin found in the liver and in the kidney.

FIG. 21: Apparent number of LSR receptors expressed in the liver ofcontrol, ob/ob and db/db mice.

FIG. 22: Effect of anti-LSR antibodies on the proportion between thequantities of ¹²⁵I-leptin distributed in the liver and in the kidney.

FIG. 23: Effect of increasing leptin concentrations on the LSR activityof rat hepatocytes in primary cultures. The results represent thedifferences in activity which are obtained between the cells incubatedwith and without oleate in the presence either of ¹²⁵I-LDL, or of¹²⁵I-VLDL.

FIG. 24: Capacity for inducing, by leptin, the LSR activity of rathepatocytes in primary culture. A. Apparent number of receptorsexpressed at the surface of the hepatocytes in the presence or in theabsence of leptin, estimated by the measurement of the quantity of¹²⁵I-LDL bound in the presence of oleate. B. Effect of cycloheximide, ofcolchicine and of cytochalasin B on the induction, by leptin, of the LSRactivity.

FIG. 25: Effect of leptin on the postprandial lipemic response incontrol (◯), ob/ob (ν) and db/db (∘) mice, reflected by the variation inthe plasma concentration of triglycerides (TG) after ingestion of ahigh-fat meal, with (B) and without (A) injection of murine recombinantleptin.

FIG. 26: Effect of leptin, in the presence and in the absence oflactoferrin, on the postprandial lipemic response of ob/ob mice,expressed by the measurement of the plasma concentration oftriglycerides (TG) after ingestion of a high-fat meal.

FIG. 27: Effect of leptin injection on the apparent number of LSRreceptors expressed in the liver of ob/ob and db/db mice.

FIG. 28: Postprandial lipemic response and LSR activity in control(C57BL6), ob/ob and db/db mice. A—Weight of control, ob/ob and db/dbmale mice. B—Postprandial lipemic response in control, ob/ob and db/dbmice. C—Apparent number of LSR receptors estimated by measurement of thebinding of LDL and expressed in arbitrary unit by comparison with the5′-nucleotidase activity in each plasma membrane preparation. D—Northernblot on an extract of liver total RNA. GAPDH is used as control.

FIG. 29: Effect of a long-term treatment by leptin on ob/ob mice.A—Weight change over 30 days B—Postprandial lipemic response on the 29thday of treatment C—Apparent number of LSR receptors on day 30, estimatedby the measurement of the binding of LDL and expressed in arbitrary unitby comparison with the 5′-nucleotidase activity in each plasma membranepreparation D—Northern blot analysis of the expression of LSRestablished on a total extract of liver RNA. GAPDH and actin are used ascontrols.

FIG. 30: Effect of the oleates on the binding and internalization of the¹²⁵I-LDL in normal human fibroblasts, under normal conditions.

FIG. 31: Effect of increasing concentrations of leptin on the LSRactivity of human fibroblasts HF (familial hypercholesterolemia).

FIG. 32: Inhibitory effect of antibodies directed against anNH₂-terminal (ν) or COOH-terminal (◯) peptide of gC1qR, or of controlantibodies (∘) on the LSR activity of plasma membranes of rathepatocytes, expressed as a percentage of the quantity of ¹²⁵I-LDL boundin the absence of antibodies.

FIG. 33: Effect of increasing concentrations of C1q on the binding,internalization and degradation of ¹²⁵I-LDL on rat hepatocytes inprimary culture, in the presence (ν) or in the absence (∘) of oleate.

FIG. 34: Effect of 25 ng/ml of recombinant AdipoQ on the LSR activity ina primary culture of rat hepatocytes.

FIG. 35: Effect of two successive injections of 1 mg of AdipoQ on thepostprandial lipemic response in rats after ingestion of a high-fatmeal.

FIG. 36: Effect of an intraperitoneal administration of AdipoQ for 3days on the weight and the concentrations of plasma triglycerides inrats on a normal diet or on a fatty diet.

FIG. 37: Effect of a daily injection of 100 μg of AdipoQ over 5 days, onfood intake in ob/ob and db/db obese mice.

EXAMPLES

Experimental Procedures

Materials

Na¹²⁵I is provided by Amersham (Les Ulis, France). Oleic acid, bovineserum albumin (A 2153) (BSA) and Triton X100 are obtained from Sigma (StQuentin Fallavier, France). Human lactoferrin (Serva) and sodium heparinare provided by Biowhittaker (Fontenay sous Bois, France) and Choaylaboratories (Gentilly, France) respectively. The enzymatic kits for thedetermination of triglycerides (TG) are obtained from BoehringerMannheim (Meylan, France). Suramin sodium is obtained from CBC Chemicals(Woodburg, Conn.). Dulbecco's modified Eagle medium (DMEM), trypsin andfoetal calf serum are provided by Life Technologies, Inc. (Eragny,France).

Animals

The mice C57BL/6J of the wild type, C57BL/6J ob/ob, C57BL/Ks of the wildtype and C57BL/Ks db/db are obtained from R. Janvier Breeding Center (LeGenest St Isle, France).

Cells

Normal fibroblasts (GM08333) and HF (GM00486A, GM007001B, GM00488C) areprovided by the NIGMS human genetic mutant cell repository (Camden, NJ).The cells were plated on Petri dishes of 36 mm as described above(300,000 normal fibroblasts per well, 150,000 HF fibroblasts per well),and are cultured in a humidified CO₂ incubator, in DMEM mediumcontaining 10% (normal fibroblasts) or 20% (HF fibroblasts) foetal calfserum, 2 mM glutamine, 100 U/ml of penicillin and 100 U/ml ofstreptomycin.

The hepatocytes in primary culture are obtained according to theprocedure described above (Mann et al., 1995). The cells are then platedat 900,000 cells per well or 22×10⁶ cells per flask of 165 cm². Thecells are used for the studies after 48 hours in culture.

Preparation and Radiolabelling of the Lipoproteins

The VLDLs (d<1.006 g/ml) and LDLs (1.025<d<1.055 g/ml) are isolated bysequential ultracentrifugation of fresh plasma from volunteers (Bihainand Yen, 1992; Goldstein et al., 1983) and used before 2 weeks. Thelipoproteins are radioiodinated (Bilheimer et al., 1972) and used lessthan one week after the labelling. ¹²⁵I-LDL and ¹²⁵I-VLDL are filtered(0.22 μm membrane, Gelman) immediately before use.

Preparation and Radiolabelling of Mouse Recombinant Leptin

The leptin cDNA is obtained from the mRNA of adipose tissue of the mouseC57BL/6J by PCR. The 5′ PCR primer introduces an initiation codon afterthe signal sequence which is deleted and a sequence encoding ahexahistidine end. The modified sequence encoding murine leptin iscloned into an expression vector pSE280 (Invitrogen, France) andexpressed in E. coli The sequencing of the plasmid DNA confirms thecoding sequence. The bacteria are cultured at 37° C. and the synthesisof the protein is induced by 1 mM isopropyl $-D-thiogalactopyranoside.The bacteria, recovered after gentle centrifugation, are lysed byfreeze-thaw and the DNA is digested with a deoxyribonuclease 1. Thecellular membranes are extracted with the aid of a detergent and theinclusion bodies are separated after centrifugation. After 3 washes in1% sodium deoxycholate in PBS, the inclusion bodies are solubilized in a6 M guanidine HCl solution. The renaturation of the recombinant proteinis achieved by diluting 1/100 in PBS. The renatured protein is thenpurified and concentrated on a nickel-based chelate metal affinitychromatography column (Probond, Invitrogen). The elution is carried outwith imidazole. The purity of the recombinant leptin is controlled bySDS-PAGE electrophoresis and its activity by the evaluation of satietyin mice C57BL/6J ob/ob after intraperitoneal injection of 25 μg ofleptin. The recombinant leptin is then radiolabelled using lodobeads(Pierce) and according to the method recommended by the manufacturer.

Cloning of the AdipoQ mRNA. Production and Purification of RecombinantAdipoQ Proteins

Cloning of the cDNA into an Expression Vector

Mouse adipose tissue is obtained from C57BI/6J mice and the mRNA isextracted with the aid of polydTs bound to magnetic beads (Dynabeads,Dynal, France). A cDNA library is constructed from mouse adipose tissueby reverse transcription at 40° C. using a commercial kit (SuperscriptLife Technologies) using the supplier's instructions. The cDNA specificfor AdipoQ is amplified using the following two primers:

-   5′ CTACATGGATCCAGTCATGCCGAAGAT 3′ (SEQ ID 37)-   5′ CGACAACTCGAGTCAGTTGGTATCATGG 3′ (SEQ ID 38).    The amplification product is then digested with the restriction    enzymes BamHI and XhoI and inserted into an expression vector pTRC    HisB (Invitrogen, France) at the corresponding sites. The B version    of pTRC allows the expression of heterogeneous sequences downstream    of a hexahistidine peptide which carries a recognition site for an    enterokinase and an epitope for the anti-Xpress antibody.    Bacterial Transfection and Checking of the Construct

The plasmid thus obtained is transfected into E. coli D115 α.Furthermore, the DNA of the plasmid is extracted and the heterologousinsert is sequenced.

Cell Culture, Extraction and Purification of the Recombinant Protein

The recombinant bacterial cells are cultured at 37° C. in an LB mediumcontaining antibiotics until the OD at 600 nm reaches 0.2. Theproduction of recombinant protein is then induced by adding 1 mMisopropyl-β-D-thiogalactopyranoside to the culture medium. The bacterialculture is continued for 16 h at 37° C. The cells are recovered bycentrifugation. The cells are lysed using lysozyme in a Tris buffer pH7.4 in the presence of NaCl, PMSF and sodium deoxycholate. The DNA isdegraded by sonication. After centrifugation, the recombinant protein isseparated from the supernatant using a Probond column (Invitrogen,France). This column contains charged nickel which has affinity for thehexahistidine peptides. The elution is carried out in the presence ofimidazole. The protein concentration is estimated by the Lowry methodafter having dialysed the product of the elution. The purity of theprotein obtained is tested by SDSPAGE electrophoresis, which shows asingle band.

Example 1 Identification of the Protein Complex Responsible for the LSRActivity: Partial Purification and Characterization By Means ofPolyclonal Antibodies

The technique of ligand blotting was used to identify the proteincomplexresponsible for the LSR activity. This technique, described in detail byMann et al., 1995, is detailed below.

Ligand Blotting

The technique consists in isolating, by differential centrifugation(Belcher et al., 1987) rat liver membranes, and in solubilizing themembrane proteins in a solution containing 125 mM octylglucoside, 20 mMTris and 2 mM EDTA, pH 8. The proteins thus solubilized are separatedunder nondenaturing conditions on a preparative SDS gel (thickness 5 mm)consisting of a gradient from 4 to 12% polyacrylamide (35-50 mg ofprotein per gel). For part of the gel, the proteins are thenelectrotransferred (semi-dry transfer, 21 V, 45 min, Biorad) onto anitrocellulose membrane. After blocking the free sites of this membranein a PBS solution containing 3% albumin, the membrane is incubated with40 μg/ml of ¹²⁵I-LDL in the presence (FIG. 8, lane 2) or in the absence(FIG. 8, lane 1) of 0.8 mM oleate. The membrane is then washed fivetimes for 10 minutes in PBS containing 0.5% (v/v) Triton X100, andexposed on a Phosphor Imager screen.

Analysis of the image obtained in the presence (FIG. 8, lane 2) or inthe absence (FIG. 8, lane 1) oleate shows the presence of 3 main bandswhich have bound the LDLs. The apparent MW of the first band is about240 kDa, that of the second is 115 kDa and that of the third is 90 kDa.On the basis of these observations, two hypotheses are formulated: onthe one hand, the LSR activity is linked to the presence of severaldistinct proteins; on the other hand, the same type of image can beexplained by a multimeric organization of a protein complex.

In order to check this hypothesis, the inventors undertook thepurification of the band having the highest apparent molecular weight(240 kDa). The partial purification of this protein, designated “bandA”, is carried out by preparative electrophoresis as follows.

Partial Purification of LSR

The technique consists in isolating, by differential centrifugation(Belcher et al., 1987) rat liver membranes, and in solubilizing themembrane proteins in a solution containing 125 mM octylglucoside, 20 mMTris and 2 mM EDTA, pH 8. The proteins thus solubilized are separatedunder nondenaturing conditions on a preparative SDS gel (thickness 5 mm)consisting of a gradient from 4 to 12% polyacrylamide (35-50 mg pergel). For part of the gel, the proteins are then electrotransferred(semi-dry transfer, 21 V, 45 min, Biorad) onto a nitrocellulosemembrane. After blocking the free sites of this membrane in a PBSsolution containing 3% albumin, the membrane is incubated with 40 μg/mlof ¹²⁵I-LDL in the presence (FIG. 8, lane 2) or in the absence (FIG. 8,lane 1) of 0.8 mM oleate. The membrane is then washed five times for 10minutes in PBS containing 0.5% (v/v) Triton X100, and exposed on aPhosphor Imager screen. The proteins of interest are electroeluted(Eletroeluter, Biorad).

The rat liver plasma membrane proteins were prepared and separated on apolyacrylamide gel as above. The precise location of band A wasestablished by ligand blotting carried out after electrotransfer ofpreprative gel sample removed at various levels.

The gel fragments containing band A are then collected, electroelutedand concentrated (speedvac), and then tested for their capacity to bindthe LDLs in the presence of oleate after electrophoresis and transferonto nitrocellulose membranes (FIG. 8, lane 3; 80 μg of protein/lane).

The proteins thus obtained were also used to produce polyclonalantibodies whose specificity was tested by Western blotting (FIG. 8,lane 4).

Preparation of Polyclonal Antibodies

The LSR proteins used as antigens for the production of anti-LSRantibodies were prepared as indicated above.

The antigen preparation is injected subcutaneously into a rabbit in thepresence of complete Freund's adjuvant, followed by a conventionalimmunization procedure. The titer of the antibody directed against therat proteins is determined regularly (dot-blot technique). When thelatter is judged to be sufficient, the specificity of the antibodiesobtained is tested by Western blotting on a preparation of solubilizedproteins of rat liver membranes as described above, with anti-rabbit IgGgoat antibodies labelled with iodine I¹²⁵ as second antibodies.

The Western blot results after electrophoresis under nonreducingconditions indicate that the antibodies produced from the proteins ofband A bind to 3 main protein bands (240 kDa, 115 kDa and 90 kDa) whichbind the ¹²⁵I-LDL in the presence of oleate (FIG. 8, lane 4). To verifythe link between these protein complexes and the LSR activity, theeffect of these polyclonal antibodies on the LSR activity was tested.

The methods used are described in detail below (Mann et al., 1995;Troussard et al., 1995). The LSR activity is estimated by measuring thebinding of lipoproteins to plasma membranes and by measuring thebinding, internalization and degradation of the lipoproteins on primarycultures of rat hepatocytes.

Measurement of the Binding of Lipoproteins on Plasma Membranes

The LSR activity is measured on a preparation of rat liver plasmamembranes (Bartles and Hubbard, 1990). These membranes exhibit 10 to15-fold enrichment with 5-nucleotidase (marker specific for plasmamembranes). 100 μg aliquots of proteins are incubated for 30 minutes at37° C. in the presence or in the absence of 0.8 mM oleate in a finalvolume of 250 μl supplemented with 100 mM PBS, 2 mM EDTA, 350 mM NaCl,pH 8 (buffer A). The oleate is added in a volume of 5 to 10 μl ofisopropanol. The excess and unbound oleate is then removed by 6 washes.The pellets are resuspended in 250 μl of incubation buffer, sonicatedfor 5 seconds, power 1.90% in the active cycle, and then centrifuged for15 min at 18,000 rpm. The activated membranes are incubated for 1 hourat 4° C. with various concentrations of antibody and then with 5 μg/mlof ¹²⁵I-LDL (1 hour at 4° C.). 25 μl of 2% BSA are added to theincubation mixture. The quantity of ¹²⁵I-LDL bound to the membranes ismeasured by sedimenting the membranes by centrifugation after havingdeposited 200 μl of the incubation mixture on a layer of 5% (WN) of BSAin buffer A. The supernatants are removed by aspiration, the tubebottoms are cut off and their radioactivity is counted in a γ counter.

The inhibitory effect of anti-LSR antibodies on the LSR activity,compared to that of any preparation of rabbit immunoglobulins is shownin FIG. 9A. The inhibition of the LSR activity by the anti-LSRantibodies confirms that the multimeric complex described above isresponsible for the activity of the receptor and validates the ligandblotting technique used to identify it. The effect of the anti-band Aantibodies was, in addition, tested on the other steps of the activityof the receptor: the internalization and the degradation of lipoproteinsby the LSR expressed at the surface of hepatocytes in primary cultures.

Measurement of the Binding, Internalization and Degradation ofLipoproteins by Hepatocytes

The LSR activity in the primary cultures of rat hepatocytes is measuredby the binding, internalization and degradation of I¹²⁵I-LDL and¹²⁵I-VLDL (LDL: low-density lipoprotein; VLDL: very low-densitylipoprotein), as described in Bihain and Yen, 1992 and Mann et al.,1995.

To measure the effect of the anti-LSR antibodies on the binding,internalization and degradation of LDLs by LSR, primary cultures of rathepatocytes (48 h after plating) are incubated in the presence of 20 ngof leptin/well for 30 min at 37° C., followed by the addition ofanti-LSR antibodies in the presence or in the absence of oleate. Afterincubating at room temperature for 30 min, ¹²⁵I-LDL (20 μg/ml) is addedand then the cells are incubated for 4 h at 37° C. The binding,incorporation and degradation of ¹²⁵I-LDL are measured as described inBihain and Yen, 1992 and Mann, et al., 1995.

The data in FIG. 9B show that the anti-band A antibodies inhibit most ofthe activity of binding of the LDLs to the LSRs present at the level ofthe hepatocytes. This inhibition induces a decrease in the sameproportions in the internalization and proteolytic degradataion of thelipoproteins.

The anti-band A antibodies are thus characterized as anti-LSR. Theirrelative specificity was defined by a selective immunoprecipitationmethod. Extracts of hepatocytes in primary culture areimmunoprecipitated by means of the anti-LSR antibodies described above,according to the protocol described below.

Immunoprecipitation of Extracts of Hepatocytes in the Presence ofSpecific Antibodies

Primary cultures of rat hepatocyte (Oukka et al., 1997) are incubatedfor 60 minutes to 2 hours in the presence of a mixture of ³⁵S-methionineand ³⁵S-cysteine (Promix, Amersham). This medium is then removed and thecells are washed and then incubated in PBS containing 1% of Triton X100.This cellular lysate is then incubated in the presence of non-specificantibodies and then of protein A. The equivalent of 40 μg of specificanti-LSR antibodies is then added and the LSR-antibody complexes areprecipitated with the aid of a second preparation of protein A. Afterwashing, the complexes are dissociated in the presence of 1% SDSsupplemented or otherwise with 5% β-mercaptoethanol, incubated at 100°C. for 5-10 minutes, and separated on a 10% acrylamide gel. The gels aredried and exposed on a Phosphor Imager screen. Each of the lanescontains the equivalent of a 165 cm² flask, that is to say 22×10⁶ cells.

Analysis of the immunoprecipitation results indicates that undernonreducing conditions (FIG. 10, lanes 2—without incubation at 100°C.—and 3—with incubation at 100° C.—), the antibodies reveal 3 principalprotein bands: 2 of apparent molecular weight 240 kDa and 180 kDa, 1 ofapparent molecular weight 68 kDa. The presence of 2 bands of weakerintensity, corresponding to a molecular weight of 115 kDa and 90 kDa,can also be noted. This experimental approach therefore essentiallyidentifies the same protein elements as those identified by the ligandblotting method. It can be observed, moreover, that under reducingconditions (FIG. 10, lanes 1 and 4), the elements of high molecularweight dissociate into 3 elements of apparent molecular weight 68 kDa,56 kDa and 35 kDa, respectively.

The relative intensity of the 68 kDa and 56 kDa bands is similar whereasthat of the 35 kDa band is about ¼ of that of the other two.

Example 2 Cloning of the c-DNA Encoding the α- and β-LSR

The screening of an expression library by means of the anti-LSRantibodies described above was carried out as indicated below.

Screening of an Expression Library

After infection of bacteria with lambda GT11 bacteriophages containingrat liver cDNA (commercially obtained from Clontech Laboratories Inc.)(5′ Strech Plus c-DNA Library), the cells are plated on LB MgSO₄ medium.After 4 hours of culture at 42° C., a nitrocellulose membrane,previously incubated in a 10 mM IPTG solution, is deposited in the Petridishes. Four hours later, the first membrane is removed and a second isapplied to the Petri dish.

Each membrane is immersed in a Petri dish containing blocking bufferkept stirring for one hour. Next, the antibody is added to a finalconcentration of 10 g/ml of blocking buffer (Huynh et al., 1984; Youngand Davis, 1983a and 1983b). The membranes are then washed three timesfor 10 minutes with TNT (10 mM Tris, 150 mM NaCl, 0.05% Tween 20).

The membranes are incubated in the presence of secondary antibodies(alkaline phosphatase-conjugated affinipure F(ab′)2 fragment goatanti-rabbit IgG; Immunotech) at a final concentration of 0.08 μg/ml ofblocking buffer (TNT+ 5% powdered skimmed milk, Pâturage trademark).

After washing the membranes in TNT, they are incubated in the presenceof BCIP (5-bromo-4-chloro-3-indolyl phosphate) and of NBT (nitro bluetetrazolium) until a colour is obtained.

The positive clones are then recovered on the dishes, titrated andsubjected to the same immunoscreening procedure so as to confirm thatthey are true positives (secondary screening). Optionally, a tertiaryscreening may be carried out. The phage DNA of the selected clones,isolated from a bacterial lysate (Clontech protocol), and digested withthe restriction enzyme EcoR1 is inserted at the EcoR1 site of theplasmid pBluescript SK+.

Two clones containing an insert of 1.8 kb were thus obtained, and provedto be of identical sequences. The hybridization of rat liver mRNA (2 μgof polyA+ mRNA) with a probe corresponding to the BgIII-XbaI fragment ofthis insert revealed two bands of sizes 1.9 kb and 2.1 kb (FIG. 11A)respectively. Northern blot analysis, with a probe corresponding to theXbaI-XbaI fragment of this insert, of the tissue distribution of thecorresponding messengers showed that they are preferably expressed inthe liver (FIG. 11B). The Northern blotting was carried out according tothe following protocol.

Northern Blotting

The membranes containing the mRNAs of different rat tissues (Clontech)were hybridized with fragments of the cDNA for the LSR gene and of thecDNA for human β-actin (Clontech), labelled with [³³P]dCTP, in 5×SSPE,10× Denhardt buffer containing 0.5% SDS, 100 μg/ml of salmon sperm DNA,50% deionized formamide, at 42° C. for 16 hours. The membranes were thenwashed in 2×SSC, 0.5% SDS at room temperature and in 1×SSC, 0.1% SDS at65° C., and then exposed on the Phosphor Imager (Molecular Dynamics).

A cDNA corresponding to the 1.9 kb band was reconstructed by 5′RACE PCRfrom the 1.8 kb fragment and sequenced.

In order to elucidate the presence of multiple bands in Northernblotting, several pairs of primers defining fragments of a rat cDNAsequence were synthesized and used as primers for a PCR amplification(FIG. 11C). The sequences of the oligonucleotides used are listed below:

a: 5′-GTTACAGAATTCGCCGCGATGGCGCCGGCG-3′ (SEQ ID 20) b:5′-GCCAGGACAGTGTACGCACT-3′ (SEQ ID 21) c: 5′-ACCTCAGGTGTCCCGAGCAT-3′(SEQ ID 22) d: 5′-GAAGATGACTGGCGATCGAG-3′ (SEQ ID 23) e:5′-ACCTCTATGACCCGGACGAT-3′ (SEQ ID 24) b′: 5′-CACCACCCTGACAGTGCGTA-3′(SEQ ID 25) c′: 5′-CTGGGGGCATAGATGCTCGG-3′ (SEQ ID 26) d′:5′-GCCCTGGAAGGCCTCGATCG-3′ (SEQ ID 27) e′: 5′-CAAGTCCCTAGGATCGTCCG-3′(SEQ ID 28)

Whereas each pair of primers shows a single fragment, the bc′ pair makesit possible to amplify three fragments of different sizes. Analysis ofthe sequences of these fragments makes it possible to reconstitute thesequence of three complete cDNAs for rat LSR, having sizes of 2097 bp(SEQ ID 1), 2040 bp (SEQ ID 3) and 1893 bp (SEQ ID 5) respectively, andall three corresponding to the same precursor messenger by alternativesplicing.

These three cDNAs contain an open reading frame starting with an AUGcodon at position 219 surrounded by a Kozak consensus sequence (Kozak,1987 and 1990). The predicted molecular weights of the proteins encodedby these three cDNAs are 66 kDa, 64 kDa and 58 kDa, respectively.

The two cDNAs encoding respectively the longest and the shortest formsof rat LSR were then translated in vitro as indicated below.

Translation in Vitro

The cDNAs are subcloned into the plasmid pcDNA3; transcription andtranslation in vitro are carried out using the Promega TNT kit. Theproducts of translation, labelled with ³⁵S-methionine and ³⁵S-cysteine,are visualized after electrophoresis on a polyacrylamide gradient gel(10%) and exposure on Phosphor Imager.

The molecular weights of the products obtained, that is to say 68 kDaand 56 kDa (FIG. 12), correspond closely to those of the α and βsubunits of LSR.

To define if the products of these mRNAs are responsible for thereceptor activity, three different experimental approaches were used.

Firstly, two peptides corresponding to residues 169-186(SAQDLDGNNEAYAELIVLGR: SEQ ID 29) of the LSR produced from the mRNA ofsize 2097 bp and to residues 556-570 (EEGQYPPAPPPYSET: SEQ ID 30) weresynthesized. The sequence of these peptides is common to the threeproteins identifed above. Antibodies directed against these syntheticpeptides were obtained according to the protocols indicated above. FIGS.13C and 14C show that these anti-LSR peptide antibodies have aninhibitory effect on the binding of the LDLs to the LSRs present on ratplasma membranes, measured according to the protocol described inExample 1.

Secondly, a partial purification of the α and β subunits was obtained byselective solublization with the aid of sarkosyl; a study using Westernand ligand blotting showed that the α and β components bind the anti-LSRpolyclonal antibodies (FIG. 13B, lane 1), the anti-LSR peptideantibodies (FIG. 13B, lane 2 and FIG. 14B, lane 2), and the LDLs afterincubation with oleates (FIG. 13B, lane 4). Ligand blotting was carriedout according to the protocol described in Example 1; Western blottingwas carried out as indicated below.

Western Blotting

Primary cultures of rat hepatocytes are prepared as indicated in“Experimental procedures” The cells harvested after 48 hours of cultureare washed and lysed in PBS containing 1% Triton ×100. The lysates aredeposited on a 10% SDS-PAGE gel under reducing conditions (2% SDS, 5%β-mercaptoethanol and 20 mM DTT, at 56° C. for 1 h). After transferringonto a nitrocellulose membrane, the Western blotting is carried out withIgG antibodies directed against the LSR receptor.

Thirdly, the labelled proteins LSR 66 and 58 obtained by in vitrotranslation from the cDNAs LSR-Rn-2097 and LSR-Rn-1893 are used toestimate the effect of oleate on the binding of the LDLs according tothe protocol detailed below.

Binding of the LDLs onto the LSR Proteins Expressed in Vitro(“Flotation”)

The ³⁵S-cysteine or ³⁵S-methionine labelled products of translation invitro (17 μl) are incubated for 1 hour at 37° C. in the presence of 100μg/ml of LDL, 1 mM oleate in buffer A, in a final volume of 400 μl. Anequal volume of 8% (w/v) BSA is added. The density is adjusted to 1.21g/ml (assuming an initial density of 1.025 g/ml), with sodium bromide.The samples are then deposited on a sodium bromide solution at 1.063g/ml, and then centrifuged for 20 hours at 4° C. (Beckman SW41 rotor). Avolume of 1 ml is collected at the surface, dialysed againstelectrophoresis elution buffer, and the radioactivity is counted(Beckman β counter).

Oleate increases the binding of LDL to LSR 56 (respectively LSR 68) by afactor of 2 (5 respectively). It can thus be shown that the α and βsubunits of rat LSR, encoded respectively by the cDNAs LSR-Rn-2097 andLSR-Rn-1893 (LSR 56 and LSR 68), preferably bind the LDLs afterincubation with oleate.

All these results indicate that the cDNAs LSR-Rn-2097 and LSR-Rn-2040encode two proteins which are indistinguishable by electrophoresis andwhose apparent molecular weight is 68 kDa; these proteins correspond tothe band comprising the α and α′ subunits of LSR, which is identifiedafter immunoprecipitation under reducing conditions. The β subunit ofLSR is presumably the product of translation of the cDNA LSR-Rn-1893.The analyses of stoichiometry after immunoprecipitation indicate thatthe multimeric complex of apparent molecular weight 240 kDa is theresult of an assembly of an α subunit with three β subunits. Analysis ofthe various domains of the proteins corresponding to the α and β-LCRs iscompatible with a lipoprotein receptor function.

Example 3 Analysis of the Activity of a Recombinant LSR Receptor, andits Subunits, in Transfected Cells

The inventors also expressed a recombinant LSR receptor in CHO cellsaccording the following protocol.

Transfection with cDNA Sequences Encoding the LSR Receptor

In order to study the activity of each of the recombinant subunits ofLSR, as well as the activity of a reconstituted receptor, the inventorsused the expression plasmid pcDNA3 (No et al., 1996) to study theexpression, in animal cells, of either cDNA encoding the α subunit (αplasmid), or of a cDNA encoding the β subunit (β plasmid), of rat LSR.The LSR cDNAs were subcloned into the plasmid pcDNA3 (Invitrogen) usingthe EcoRI and/or NotI restriction sites. Once obtained, these constructsare used to transfect CHO (Chinese hamster ovary) animal cells.

After 48 hours of culture, CHO (Chinese hamster ovary) cells (CHO-K1,CCL-61, ATCC, Rockville, Md.) were distributed into 6-well plates(Falcon) at 2.5-2.75×10⁵ cells/well. After 24 h of culture in a Ham F-12medium containing 10% (v/v) FBS, 2 mM glutamine and 100 units/ml ofpenicillin and streptomycin, a maximum of 2 μg of plasmid/well weretransfected using Superfect (Qiagen) according to the supplier'sinstructions (10 μl Superfect/well, 2 h at 37° C. in a Ham F-12 mediumfree of serum). The plates were then washed in PBS in order to removethe transfection reagents and the cells were then cultured in a Ham F-12medium containing serum. The LSR activity was measured 48 h aftertransfection according to the protocols detailed in Example 1.

The inventors tested the effect of a co-transfection with the α and βplasmids compared with that of a transfection with the α plasmid alone,or with the β plasmid alone, on the three stages of the activity of theLSR receptor according to the protocols detailed below. FIGS. 15 and 16show the comparisons between the LSR activities obtained on therecombinant cells expressing the α subunit alone, or the two α and βsubunits; similar results are obtained for the β versus α+β comparison,which is compatible with the comparative analysis of the primarysequences of each of the subunits (each of them also carrying thepotential binding sites for lipoprotein ligands and fatty acids, such asoleate).

Effect of a Transfection With the LSR (α) Plasmid Alone, or of aCo-transfection with the LSR (α) and LSR (β) plasmid, on the binding,Internalization and Degradation of the LDLs

The CHO-K1 cells were transiently transfected with increasingconcentrations of α plasmid and co-transfected with 0.4 μg of α plasmidand increasing concentrations of β plasmid. After 48 h of culture, thecells were washed once with PBS and incubated for 3 h at 37° C. with 20μg/ml ¹²⁵I-LDL in the presence or in the absence of 1 mM oleate in DMEMcontaining 0.2% BSA, 5 mM Hepes, and 2 mM CaCl₂, pH 7.5. Next, the cellswere washed as described above and incubated at 4° C. for 1 h with 10 mMsuramin in PBS.

To measure the binding of the LDLs (FIG. 15), the medium was recoveredand passed through a γ counter in order to evaluate the quantity ofbound ¹²⁵I-LDL. The results are the mean values of two measurements. Forthe measurement of the internalization and the degradation of LDLs (FIG.16), the quantity of ¹²⁵I-LDL internalized and degraded was measuredaccording to the protocols detailed in Example 1.

The co-transfection with α and β plasmids makes it possible to establishthree stages of LSR activity (FIGS. 15 and 16).

The inventors also observed that the co-transfection with the α and βplasmids increases the LSR activity compared with a transfection withonly an α plasmid. The results suggesting a more efficient activity ofthe LSR when the ([β]/[α]) ratio between the concentrations of β and αsubunits expressed increases, is compatible with the observation thatthe LSR receptor might consist of the assembly of an α (or α′) subunit,and of several, probably three, β subunits.

The results show that only the co-transfection of the β and α subunitsallows the overexpression of a completely functional LSR receptor in thesense that it allows the complete proteolytic degradation of theprotein.

In order to characterize the lipoprotein degradation activity obtainedabove in cells transfected with the LSR cDNAs, the inventors finallytested the capacity of anti-LSR antibodies to inhibit the binding ofLDLs as measured above, as well as the substrate-specificity thereof.

Characterization of the Lipoprotein Degradation Activity Obtained inTransfected Cells Expressing a Recombinant LSR Receptor

The CHO cells were transfected with the α end β plasmids in aconcentration ratio of 1 to 3.

FIG. 17A shows that the LDL binding activity obtained in the transfectedcells (expressed relative to the same activity observed innontransfected control cells) is specifically inhibited by the anti-LSRantibodies.

FIG. 17B shows the LDL binding activity obtained in the cellstransfected in the presence of various nonlabelled lipoproteins actingas competitive ligands. The results show a ligand specificity similar tothat observed for the endogenous LSR activity in rats (Mann et al.,1995): the rat chylomicrons are the preferred substrates for the ratrecombinant LSR; then come in particular, in decreasing order ofspecificity, the VLDLs and then the LDLs.

Example 4 Involvement of LSR in the Clearance of Cytokines

The analysis of the sequence of the α subunit of LSR reveals acysteine-rich region which corresponds to a Tumor Necrosis Factor typecytokine receptor signature. LSR is, however, distinguishable from thecytokine receptors by the presence of signals allowing rapid endocytosisof the receptor/ligand complex (clathrin motif).

The inventors formulated the hypothesis that this receptor could servefor the removal of cytokines, and in particular of leptin; in order toverify this hypothesis they analysed the degradation of recombinantleptin by hepatocytes in primary culture according to the protocolbelow.

Degradation of Leptin by Hepatocytes in Primary Culture

Primary cells of rat hepatocytes are incubated for 4 hours at 37° C.with 20 ng/ml of ¹²⁵I-leptin in the absence or in the presence of 0.5 mMoleate, 75 μg/ml of RAP, 200 μg/ml of non-specific antibodies oranti-LSR specific antibodies, or 50 μM chloroquinine. The medium is thenrecovered and the quantity of ¹²⁵I-leptin degraded is measured.

As indicated in FIG. 18, the degradation of leptin by hepatocytes inprimary culture is inhibited by:

-   a) polyclonal antibodies directed against LSR. These antibodies also    inhibit, in the same proportions, the LSR activity,-   b) the 39 kD Receptor Associated Protein (RAP); this protein blocks    the LSR activity in vitro and retards the clearance of chylomicrons    in vivo (Troussard et al., 1995; Willow et al., 1994)-   c) chloroquine; this cellular poison prevents the acidification of    the endocytosis vesicles and inhibits the activity of the lysosomal    proteases,-   d) oleate; this free fatty acid induces a change in the conformation    of LSR which unmasks the lipoprotein binding site.

This indicates that the FAF (Fatty Acid Free) conformation of LSR isprobably the only one which is compatible with the role of bindingfollowed by degradation of leptin. The non-specific immunoglobulins arewithout effect on the degradation of leptin (FIG. 18).

In order to verify the binding of leptin to LSR, the rat liver plasmamembrane proteins were deposited on an affinity chromatography columncontaining recombinant leptin, according to the protocol detailed below.

Leptin Affinity Chromatography

A Hi-trap column (Pharmacia) is used: 5 mg of leptin are bound onto 1 mlof column according to the methods recommended by the manufacturer. Theplasma membrane proteins are solubilized from rat livers as indicatedabove (Mann et al., 1995), and then dialysed overnight against PBS pH7.4, 0.1% Tween 20. The column is washed in the same buffer and theprotein extract is deposited at a rate of 0.2 ml/minute. The column iswashed with 6 ml of the same buffer. It is then eluted with the samebuffer supplemented with 100 mM glycine pH 3; 20 fractions of 500 μl arethen neutralized with 5 μl of PBS, 0.1% Tween 20, pH 8. 50 μl of eachfraction are deposited on a nitrocellulose membrane for dot-blotanalysis by means of anti-LSR antibodies. The positive fractions (1, 3,4, 7 and 8) are dialysed against 24 mM ammonium bicarbonate, 0.01% Tween20, pooled and concentrated in a Speedvac in a final volume of 300 μl.40 μl of the final product are analysed by Western blotting by means ofanti-LSR antibodies.

FIG. 19 shows that the anti-LSR antibodies specifically recognize the αsubunit which, after binding to leptin, was released by the glycinebuffer.

Experiments of stable transfection of the α subunit will make itpossible to measure the affinity of leptin for this new receptor.

All these results suggest that LSR represents one of the pathways forthe degradation and elimination of leptin. The in vivo injection ofradiolabelled recombinant leptin showed, both in the obese mice and inthe control mice, a rapid speed of clearance and a preferential captureof leptin by the liver and the kidney: 50% of the injected dose is foundafter 10 minutes in these two organs. In order to analyse the mechanismsfor the selective capture of leptin, the inventors compared thequantities of leptin and of β2 microglobulin (soluble protein having amolecular weight close to that of leptin, chosen as control) present inthe kidney and liver of normal mice and of two obese mouse lines 5minutes after injection of the same tracer dose of these tworadiolabelled proteins.

Measurement of the Clearance of Leptin in Mice

The female control, ob/ob, or db/db mice (6-8 weeks), on an emptystomach, are anaesthetized and receive via the saphenous vein aninjection of 80 ng of murine recombinant ¹²⁵I-leptin or of¹²⁵I-β₂-microglobulin (Sigma, labelled by the Iodobeads method, likeleptin). Five minutes later, the animals are infused with aphysiological saline solution (15 ml, at 4° C.). The tissues arecollected and counted for their radioactivity (Gamma counter). In somecases, an anti-LSR antibody or a control protein are injected 30 minutesafter injection of ¹²⁵I-leptin. It is important to note that thelabelling of leptin with ¹²⁵I has no effect on its biological activity.

The results presented in FIG. 20 show that the quantity of leptinselectively captured by the liver is reduced in the obese mice, comparedwith the control mice; moreover, no difference is observed between thevarious lines as regards the renal capture of leptin.

The inventors then measured the number of LSR receptors in control,ob/ob and db/db mice according to the following protocol.

Measurement of the Apparent Number of LSR Receptors on Plasma Membranes

The apparent number of LSR receptors on plasma membranes is measured aspreviously described (Mann et al., 1995) by estimating the quantity ofLDL bound to a plasma membrane preparation. The plasma membranes (100μg) are incubated with 1 mM oleate; they are then washed three times asindicated above, and then incubated for 1 hour at 37° C. with 40 μg/mlof ¹²⁵I-LDL. The quantity of ¹²⁵I-LDL bound to the plasma membranes isthen determined by counting. The mean is established on 3 measurementsper animal for 3 different animals in each of the groups.

FIG. 21 shows that the number of LSR receptors in obese animalsexhibiting either a deficiency in leptin (ob/ob), or a deficiency in theob receptor (db/db), is significantly reduced. The reduction in theselective hepatic capture of leptin in obese mice coincides with thereduction, in these animals, of the apparent number of LSR receptors.

The inventors finally tested, according to the protocol presented below,the effect of anti-LSR antibodies on the distribution of leptin betweenthe liver and the kidney, 5 minutes after injection of a tracer dose.

Measurement of the Distribution of Leptin Between the Liver and theKidney in the Presence of anti-LSR Antibodies

Control mice are anaesthetized and then they are injected intravenouslywith 1 mg of non-specific IgG antibody or of anti-LSR IgG antibody.After 30 minutes, 80 ng of ¹²⁵I-leptin are injected and, after 5minutes, an infusion of physiological saline solution at 4° C. Thetissues are removed immediately and the radioactivity is measured. Theresults represent the mean and the standard deviation obtained for 3animals for each of the groups.

As shown in FIG. 22, the hepatic capture of leptin is reduced and therenal capture is increased by the anti-LSR antibodies, compared with thecontrol immunoglobulins.

These results therefore indicate that LSR is responsible for theselective hepatic capture of leptin and that a reduction in the numberof receptors is observed in the obese animals. Such a reduction mayexplain the leptin-resistance syndrome and the increase in the plasmaconcentration of leptin which is observed in most obese human subjects.

It is also possible that the LSR receptor serves as degradation pathwayfor other cytokines, in particular those produced by the adipose tissue.The importance of Tumor Necrosis Factor α and Nerve Growth Factor willbe noted in particular. These two cytokines exert a significant slimmingeffect when they are injected into human subjects (Cytokines and theirreceptors, 1996).

Example 5 Control of the LSR Activity by Cytokines

The α subunit of the LSR receptor binds leptin and possesses potentialphosphorylation sites. This makes it a receptor which not only mediatesendocytosis, but could also serve in cell signalling.

The inventors therefore tested the hypothesis according to which leptinmodulates the activity of LSR, as described below.

Measurement of the LSR Activity of Binding, Internalization andDegradation of Lipoproteins in the Presence of Leptin

Rat hepatocytes in primary culture are incubated at 37° C. for 30 minwith an increasing concentration of leptin, and then incubated at 37° C.for 4 hours with either 50 μg/ml of ¹²⁵I-LDL (specific activity: 209cpm/ng) or 50 μg/ml of ¹²⁵I-VLDL (specific activity: 157 cpm/ng) in theabsence or in the presence of 500 μM oleate. The cells are then washedand the quantities of 1-lipoproteins bound, incorporated and degradedare measured as described above in Example 1 (Bihain and Yen, 1992). Theresults shown in FIG. 23 represent the differences obtained between thecells incubated with or without oleate. Each point represents the meanof 3 measurements. The standard deviation for each point is included inthe symbol.

The addition of increasing concentrations of leptin to hepatocytes inculture increases the binding, internalization and degradation of VLDLsand LDLs (FIG. 23).

Analysis of the Capacity for Inducing the LSR Activity by Leptin

Measurement, in the Presence of Leptin, of the Apparent Number of LSRReceptors Expressed at the Surface of Rat Hepatocytes in Primary Culture

Primary cultures of rat hepatocytes are incubated for 30 min at 37° C.in the presence or in the absence of 20 ng/ml of leptin, for 10 min at37° C. in the presence of 0.8 mM oleate. The cells are washed with PBSbuffer precooled to 4° C., and then incubated for 2 hours at 4° C. inthe presence of increasing concentrations of ¹²⁵I-LDL. The cells arethen washed, lysed and the quantity of bound ¹²⁵I-LDL is measured.

Comparative Effects of Leptin in the Presence of Cycloheximide,Colchicine and Cytochalasin B

The initial conditions are identical to those described above; afterincubation with leptin, the cells are incubated for 30 min at 37° C.with 5 μM cycloheximide, 5 μM colchicine or 2.5 μM cytochalasin B. Thecells are then incubated for 10 min at 37° C. in the presence of 0.8 mMoleate. The cells are then washed with PBS buffer precooled to 4° C.,and then incubated for 2 hours at 4° C. in the presence of 50 μg/ml of¹²⁵I-LDL. 2 measurements are carried out, and the mean results arepresented.

It is thus shown that the increase in the LSR activity by leptin isobtained through an increase in the apparent number of receptorsexpressed at the surface of the hepatocytes (FIG. 24A). This increaseresults, on the one hand, from an increase in protein synthesis (it ispartially inhibited by cycloheximide, an inhibitor of proteinsynthesis). It involves, on the other hand, the mobilization of theendocytosis vesicles by the microtubule system (it is indeed inhibitedby cytochalasin B which blocks microtubular transport) (FIG. 24B).

In order to check the in vivo effect of leptin on the LSR activity, theinventors characterized the postprandial triglyceridemic response ofcontrol, ob/ob and db/db mice after a force-fed test meal according tothe following protocols.

Measurement of the Postprandial Lipemic Response in Mice Control, ob/oband db/db mice, starved since the day before, are force-fed with a mealwhich is very high in fat [60% fat (37% saturated, 27% monounsaturatedand 36% polyunsaturated fatty acids), 20% protein and 20% carbohydrate]providing 56 kcal of energy/kg of the weight of the animal. Immediatelyafter the meal (time=0 hour), the mice are injected intravenously with200 μl of physiological saline solution. At various times, 20 μl ofblood are collected via the caudal vein in tubes containing 90 μg ofdisodium EDTA, and after separating the plasma by centrifugation, theplasma concentration of triglyceridernia is determined with the aid ofan enzymatic assay kit. Each point on the curves presented correspondsto the mean with standard deviation obtained for 3 measurements peranimal and for 3 different animals.Measurement of the Effect of Leptin on the Postprandial Lipemic Responsein Mice

The procedure is the same as above, except that immediately after themeal (time=0 hour), the mice are injected intravenously with either 200μl of physiological saline solution, or 200 μl of the same solutioncontaining 50 μg of murine recombinant leptin.

Measurement of the Postprandial Lipemic Response in Mice in the Presenceof Lactoferrin and/or Leptin

ob/ob mice, starved since the day before, are force-fed with a mealidentical to that described above. Immediately after the meal (time=0hour), the mice are injected intravenously with 200 μl of salinesolution containing either no supplement, or 0.5 μg of leptin, or 2.5 mgof lactoferrin or alternatively a mixture of 0.5 μg of leptin and 2.5 mgof lactoferrin. Blood is collected between 2 and 3 hours after the mealand the plasma concentration of triglycerides (TG) is measured. Thevalues obtained represent the mean with standard deviation obtained for4 measurements per animal and for 2 different animals [p<0.02 (ob/obcompared with ob/ob+leptin), p<0.01 (ob/ob compared withob/ob+lactoferrin), NS (ob/ob+lactoferrin compared withob/ob+leptin+lactoferrin)].

In agreement with the reduction in the number of LSR receptors observedin the obese mice, an amplification of the postprandial lipemic responsealso exists in the untreated obese mice. The administration of leptin bythe intravenous route, at the same time as the test meal, makes itpossible to reduce the postprandial lipemic response in the two obesemouse lines and in the control mice (FIG. 25).

This reduction in the lipemic response induced by leptin is suppressedby the administration of lactoferrin (FIG. 26), which blocks theactivity of LSR (Yen et al., 1994; Mann et al., 1995). This stronglysuggests that the reduction in the lipemic response is explained by anincrease in the LSR activity.

Finally, also in vivo, the administration of leptin induces an increasein the apparent number of LSR receptors expressed at the level of thesurface of the hepatocytes. This increase is significant both in theob/ob mice and in the db/db mice (FIG. 27).

Leptin and probably other cytokines are therefore regulators of theactivity of LSR. A syndrome of resistance to leptin or to othercytokines can lead to hypertriglyceridemia, which is either permanent orlimited to the postprandial phase.

Example 6 Effect of Leptin on the Expression of LSR; Therapeutic Effects

To reinforce correlation between the administration of leptin, thereduction in the postprandial lipemic response, and an enhancedexpression or activity of the LSR receptor, and to better evaluate thepossible therapeutic implications of the induction of the activity ofhepatic clearance of lipoproteins by leptin, the inventors supplementedthe preceding analysis with monitoring of the weight variation, of theLSR activity and of the expression of LSR mRNA, in control or obeseanimals treated with leptin or otherwise.

Postprandial Lipemic Response and LSR Activity in Control and Obese Mice

Control male mice (C57BL6) (n=8) and obese male mice (ob/ob, n=8—animalsdeficient in the leptin gene— and db/db, n=8—animals deficient in thegene for the leptin receptor—) (aged 17 weeks old) were weighed in orderto quantitatively establish the differences in weight between lines(FIG. 28A). The postprandial lipemic responses of the animals of eachline were measured in the absence of treatment with leptin as describedabove. The apparent number of LSR receptors expressed at the surface ofthe hepatic cells was measured on 4 animals of each line, as describedabove, and expressed in comparison with the 5′-nucleotidase activity(enzyme selectively measured at the level of the plasma membranes; Sigmakit). Finally, Northern blotting made it possible to estimate the levelof expression of the LSR receptor in three animals of each line,according to the protocol described above.

The higher postprandial lipemic response in the obese animals (FIG. 28B)is in agreement with the smaller apparent number of hepatic LSRreceptors in these same animals (FIG. 28C). Furthermore, the Northernblotting results (FIG. 28D) indicate that this reduction in the apparentnumber of LSR receptors in the obese animals is accompanied by areduction in the level of expression of the said receptor in the sameanimals. The inventors have shown that indeed, a reduction in the numberof mRNA encoding the LSR receptor is observed in the obese mice ob/oband db/db.

The inventors also studied the effect of a long-term treatment of atreatment with leptin on ob/ob mice (FIG. 29).

Effect of a Long-term Treatment with Leptin on ob/ob Mice

The ob/ob obese mice received a daily injection of either leptin, or ofan equivalent volume of sterile PBS, for 30 days. The injected doses are50 μg/animal from day 0 to day 4, 100 μg/animal from day 5 to day 17,and 150 μg/animal from day 18 to day 30.

Several Parameters Indicated Below are Measured:

-   -   the weight (FIG. 29A): the change in weight is measured for 6        animals, over the duration of the treatment;    -   the postprandial lipemic response (FIG. 29B): it is measured        according to the protocol detailed in Example 5 on three animals        in each group, on day 29;    -   the apparent number of LSR receptors (FIG. 29C): it is measured        according to the protocol detailed in Example 4 on three animals        in each group, on day 30;    -   the quantity of LSR mRNA (FIG. 23D): it is estimated by Northern        blotting as indicated in the protocol of Example 2.

The inventors thus observed a very significant loss of weight in theob/ob obese mice treated over 30 days with leptin. Furthermore, thetreatment with leptin causes a clear reduction in the postprandiallipemic response. This reduction in the postprandial lipemic response iscorrelated with an increase in the apparent number of LSR receptors atthe surface of the cells and with an increase in the quantity of mRNAencoding the subunits of the LSR receptor.

These results establish in vivo that LSR represents the limiting step inthe elimination of dietary lipids. Furthermore, the treatment of thisobesity inducing a weight loss causes an increase in the activity ofhepatic degradation of dietary lipids, and a reduction in thepostprandial lipemic response.

Example 7 Characterization of the Human LSR Receptor

Northern-blot Analysis

Nucleic probes for rat LSR were used to carry out Northern-blot analyseswith a membrane (Human Multiple Tissue Northern Blot, Clontech #7760-1)comprising human heart, brain, placenta, lung, liver, skeletal muscle,kidney and pancreas poly A RNAs. A band of about 2 kbp is detected inthe liver and in the kidney. Approximate quantification of thehybridization results indicate that LSR is expressed in the liver atleast 5 times more than in the kidney.

Cloning of the cDNA; Study of the Splicing Zone

Reverse transcription-PCR experiments on the mRNA made it possible todetermine with greater precision the size of exon 1 on the 5′ side andsplicing sites between exons 1 and 2. However, it is not certain thatthis end constitutes the start of this exon. In addition, a secondinitiation site exists in exon 1 which is more downstream from the firstand which exhibits a greater probability than the latter. The splicingbetween exons 1 and 2 was different between the human RNA and the ratRNA.

The amplification was carried out with several pairs of primers:

a: 5′-ATGCAACAGGACGGACTTGGA-3′ (SEQ ID 31) exon 1 b:5′-TCAGACGACTAAACTTTCCCGACTCAGG-3′ (SEQ ID 32) exon 10 c:5′-CTACAACCCCTACGTTGAGT-3′ (SEQ ID 33) exon 2 d:5′-TCGTGACCTGACCTTTGACCAGAC-3′ (SEQ ID 34) exon 3 e:5′-CCTGAGCTACTCCTGTCAACGTCT-3′ (SEQ ID 35) exon 6 f:5′-AGGCCGAGATCGCCAGTCGT-3′ (SEQ ID 36) exon 9

The amplification carried out with the ab pair of primers led to twoproducts 1.8 kb and 2 kb in size after separation on an electrophoresisgel. Given that the sizes of these two products can be explained by analternative splicing similar to that described in rats, the otheramplification primers were drawn. These primers made it possible toidentify the three forms of cDNA resulting from the alternative splicingof the RNA.

The first cDNA which contains the totality of the ten exons is calledLSR-Hs-2062 and corresponds to SEQ ID 7. It corresponds to the rat cDNALSR-Rn-2097. The second cDNA contains exons 1, 2, 3, 5, 6, 7, 8, 9 and10, and is called LSR-Hs-2005. It corresponds to SEQ ID 9. This cDNAcorresponds to the rat cDNA LSR-Rn-2040. Finally, the cDNA containingexons 1, 2, 3, 6, 7, 8, 9 and 10 is called LSR-Hs-1858 and its sequenceis listed in SEQ ID 11. It corresponds to the rat cDNA LSR-Rn-1893.

It should be noted that it was possible to demonstrate a slippage of thesplicing site at the boundary of exon 8. This slippage, of the tripletTAG at position 19953-19955 of SEQ ID 19 to the contiguous triplet AAGat position 19956-19958 of SEQ ID 19, results in the loss of the Gluresidue at position 386 of the cDNA of SEQ ID 8.

The sequences of the proteins encoded by the cDNA LSR-Hs-2062,LSR-Hs-2005 and LSR-Hs-1858 correspond respectively to SEQ ID 8, 10 and12. The biological protein sequences can start at the first ATG codonobserved in the reading frame (position 35 of the protein sequence).However, the preferred codon for initiation of translation is moredownstream at position 83 of the protein sequence. Furthermore, it isquite possible that this initiation codon is more upstream in the 5′region of exon 1 not yet determined or in a possible exon preceding thelatter.

Finally, FIGS. 3A and 3B represents a schematic representation of thevarious protein forms identified in humans, indicating the conservedmotifs.

This analysis makes it possible to conclude that three α, α and βsubunits of LSR, which are equivalent to the LSR 66, LSR 64 and LSR 58forms in rats, exists in humans.

Identification and Isolation of the Genomic Sequence for Human LSR

Screening of public data banks of nucleic sequences (Genebank, version:101) both with the sequence of mouse lisch7 (Accession No.: U49507) andwith that of rat LSR_(—)2097 isolated by the inventors made it possibleto isolate two human genomic DNA sequences. They are cosmids whoseaccession numbers are AC002128 and AD000684, of respective sizes 45,328bp and 41,936 bp. These two cosmids partially overlap. The 3′ end of thecosmid AC002128 overlaps, over 12838 bp, the 5′ end of the cosmidAD000684. On the common portion of 12,838 bp, the sequences are 100%identical, apart from two deletions at positions 822 and 3170 of thecosmid AD000684. The human LSR gene is distributed over the two cosmids.To facilitate the study of this region, a complete genomic sequence wasreconstituted: the 45,328 bp of the cosmid AC002128 were added to thesequence of the cosmid AD000684 between the 12,839 base and the 41,936base. The combination constitutes a sequence of 74,426 bp. A genomicsequence covering the LSR gene, was extracted (SEQ ID 19).

The putative exons of the LSR gene were determined after alignment ofthe sequence described above with the sequences of the RNAs for mouseLisch7 and rat LSR. The validity of the splicing sites on either side ofthe putative exons was verified.

Moreover, a human genomic library consisting of BACs was screened by themethods described in Chumakov et al., 1995; the clones thus isolatedwere contiged, subcloned and then sequenced in order to obtain the humangenomic sequence encoding LSR (SEQ ID 41).

The two sequences thus obtained (SEQ ID 19 and 41) carry minordifferences which are mentioned in the accompanying listings.

Example 8 LSR Activity in Humans

Primary cultures of human fibroblasts, isolated from subjects having adeletion affecting the promoter and the first exon of the LDL receptorgene, were obtained.

The incubation of these cells in the presence and in the absence ofoleate shows that the latter induces LDL binding, internalization anddegradation activity which follows a saturation kinetics (Bihain andYen, 1992). The affinity of this receptor, induced by oleate, is maximumfor the particles high in triglycerides (VLDL and chylomicrons) as wellas for triolein and phosphatidylcholine supplemented with recombinantapoprotein E. The affinity of the LDLs for the receptor is lower thanthat of the VLDLs and the chylomicrons but, however, higher than thoseof triolein and phosphatidylcholine particles not containing ApoE, orthan those of VLDLs isolated from a subject with type III hyperlipidemiaand the ApoE E_(2/2) phenotype (Yen et al., 1994).

It was also possible to measure the LSR activity in fibroblasts ofnormal human subjects (FIG. 30), according to the protocol below.

Measurement of the Binding, Internalization and Degradation of LDLs byFibroblasts.

The fibroblasts are cultured beforehand for one week as described above,except that the medium contains 20% foetal bovine serum (Goldstein etal., 1983). Next, they are incubated with increasing concentrations of¹²⁵I-LDL in the absence or in the presence of 1 mM oleate. The cells arethen washed, lysed and counted for their radioactivity.

Example 9 Effect of Leptin on the LSR Activity in Humans

The LSR activity of human fibroblasts HF (familial hypercholesterolemia)is also increased after incubation with leptin (FIG. 31), suggestingthat as in rats, LSR participates, in humans, in the clearance ofcytokines, and its activity is modulated by the latter. Thecorresponding measurements were carried out as indicated below.

Effect of Leptin on the LSR Activity on Human Fibroblasts

The fibroblasts HF are incubated for 30 minutes at 37° C. withincreasing concentrations of leptin, and then for 2 hours at 37° C. with50 μg/ml of ¹²⁵I-LDL, in the presence of 500 μM oleate. The binding,internalization and degradation of the LDLs are measured as indicated inExample 1.

Example 10 Cloning of the cDNA for Mouse LSR; Analysis of the Productsof Alternative Splicing

The cloning of the cDNA for mouse LSR was carried out using a mouseliver mRNA library. The cloning method used is the same as that for thecDNA for human LSR. The mRNAs were purified and a reverse transcriptionPCR amplification was carried out with the specific DNA primers. Theamplification fragment was cloned to a TA cloning vector (Introgene).

A study of the products of alternative splicing with primers situated inexon 2 and in exon 9 was also carried out in a manner similar to thatcarried out for the human LSR.

Three products of alternative splicing were observed: LSR-Mm-1886,LSR-Mm-1829 and LSR-Mm-1682. LSR-Mm-1886 contains all the exons from 1to 10. LSR-Mm-1829 and LSR-Mm-1682 lack exon 4 and exons 4 and 5,respectively. These three biological forms of cDNA indeed correspond towhat was observed in humans and rats. The nucleotide sequences of thecDNAs LSR-Mm-1886, LSR-Mm-1829 and LSR-Mm-1682 are illustrated in SEQ ID13, 14 and 15, respectively. The protein sequences encoded by the cDNAsLSR-Mm-1886, LSR-Mm-1829 and LSR-Mm-1682 are illustrated in SEQ ID 16,17 and 18.

Example 11 Identification of the γ Subunit of LSR

The α and β subunits of LSR were identified as indicated above. Analysisof the products of translation of the RNAs encoding these two subunitsdoes not allow the presence of a third subunit of molecular weight ≈35kDa to be explained. This subunit is detected only after reduction ofthe LSR complex (FIG. 10, lane 4).

We purified and obtained the NH₂-terminal sequence of this γ subunit.

The purification was carried out by immunoaffinity chromatographyaccording to the following procedure.

Purification of the γ Subunit of LSR

Anti-LSR antibodies (band A) are coupled to a resin [2.5 mg of IgG per3.5 ml of affi-gel Hz immunoaffinity kit resin (Biorad 153-6060)] whichis then incubated with proteins solubilized from total membranes of ratliver (20 mM Tris buffer, 2 mM EDTA, 0.125 M octyl glucoside (5×CMC), 1%inhibitor cocktail, pH=7.4: 160 mg of membrane proteins give 41.3 mg ofsolubilized proteins (SP) in a volume of 17 ml.

-   -   The incubation is carried out for 12 hours: 17 ml filled to 50        ml with 20 mM Tris buffer, 2 mM EDTA, pH 7.4 and the 3.5 ml of        resin, with rotary shaking, at room temperature. The resin is        washed with 40 ml of 20 mM Tris buffer, 2 mM EDTA, pH 7.4 and        then eluted with 20 mM Tris buffer, 2 mM EDTA, 200 mM glycine,        pH 2.5 in 30 fractions of 500 μl. The pH of each fraction is        neutralized with 100 μl per tube of 1 M Tris buffer, 2 mM EDTA,        pH 9. 50 μl of each fraction are deposited on a nitrocellulose        membrane for dot-blot analysis: incubation with anti-LSR        antibody, and then with a second antibody coupled to alkaline        phosphatase.    -   The positive fractions from 7 to 28 are pooled in pairs and        concentrated 2.5-fold in a Speedvac. Western blotting is carried        out on the pooled, concentrated and separated fractions on a 10%        PAGE-SDS gel. Bands are observed in fractions 7 to 14 (the        fractions are pooled).    -   The two pools are dialysed against 24 mM ammonium bicarbonate        and then freeze-dried in a Speedvac. The powder is taken up in        80 μl of 20 mM Tris buffer, 2 mM EDTA, 2% SDS, 3% urea, pH 7.4        and reduced in the presence of 5% β-mercaptoethanol for 30        minutes at 100° C.    -   After migration and wet transfer in 50 mM Tris, 50 mM borate on        a sequencing membrane (PVDF) at 30 mA, the membrane is stained        with amido black.

A band with an apparent MW of about 35 kDa was thus identified and sentfor sequencing according to the Edman method.

The sequence obtained is LHTGDKAFVEFLTDEIKEE. This sequence correspondsidentically to that of a protein of molecular weight 33 kDa identifiedabove as a protein of the cellular surface which binds the globularheads of C1q (gC1q-R) (Ghebrehiwet et al., 1994). A more recentobservation indicates that this potential receptor for C1q is alsolocated in the vesicles situated under the cellular surface (van denBerg et al., 1997). This protein also corresponds to a proteinpreviously identified as p34, and which combines with a lamin receptor.This receptor possesses a long NH₂-terminal segment oriented inwards inthe cell nucleus as well as 8 transmembrane domains. This receptor bindsto lamin in a manner which depends on the degree of phosphorylation.Finally, gC1q-R combines with “splicing factor 2” (Honoré et al., 1993).The lamin receptor and “splicing factor 2” have in common thecharacteristic of containing a repeated sequence of serine and arginine(RSRS) situated at the level of the NH₂, terminal segment in the case ofthe lamin receptor and at the level of the carboxy-terminal segment inthe case of SF2.

It is remarkable to observe that both α-LSR and β-LSR exhibit repeatedsegments high in serine and arginine (FIG. 1). Our hypothesis is thatthe γ-LSR protein represents a molecular chaperone which combines withthe α and β subunits of LSR via their RSRS domain.

In order to verify this hypothesis, we obtained polyclonal antibodiesdirected against two synthetic peptides whose sequence was situated atthe carboxy- or NH₂-terminal end of the gC1q-R protein:

-   -   NH₂-terminal peptide of gC1q-R: LRCVPRVLGSSVAGY* (amino acids 5        to 19 of gC1qR) (SEQ ID 39)    -   COOH-terminal peptide of gC1q-R: C*YITFLEDLKSFVKSQ (amino acids        268 to 282 of gC1q-R) (SEQ ID 40).    -   *: amino acids differing from the protein sequence, so as to        optimize the antigenicity of the peptides.

FIG. 32 shows these antibodies specifically inhibit the activity of LSR.The antibody directed against the COOH-terminal end appears to be themost effective. These results indicate that gC1q-R, or one of itsstructurally similar homologues, represents a molecular chaperonenoncovalently combined with the LSR multimeric complex.

Example 12 Regulation of the LSR Activity by C1q and its Homologues

It has been shown that gC1q-R could bind the globular head of complementfactor 1. We sought to use this property of C1q to displace gC1q-Rcombined with LSR, and we measured the effect of increasing doses of C1qon the binding, internalization and degradation of the LDLs byhepatocytes in primary culture. FIG. 33 shows an increase in the captureand degradation of LDLs induced by human C1q, even in the absence ofoleate.

A less substantial, but nevertheless significant, increase is alsoobserved in the presence of oleate. However, under these conditions, themaximum effect is obtained for lower concentrations of C1q.

It therefore appears that gC1q-R exerts on LSR an inhibitory effectwhich is comparable to that induced by the 39 kD RAP for LRP, the LDLreceptor and LSR (Troussard et al., 1995). The displacement of thechaperone gC1q-R using its capacity to bind to complement C1q makes itpossible to lift the inhibitory effect. Analysis of the gC1q-R sequenceshows that it may be a typical membrane receptor. Indeed, the proteinpossesses no hydrophobic sequence capable of crossing the phospholipidbilayer.

The effect of complement C1q on the activity of LSR opens majorperspectives in the context of the genetics of obesity. It is possible,indeed, that mutations affecting either the gene for C1q, that forgC1q-R, or alternatively that for their analogues such as for exampleAdipoQ, cerebellin, collagen alpha 1-10, SPA and SPD (pulmonarysurfactant proteins), mannan-binding protein, and the scavenger receptoror its homologue LRP (Hu et al., 1996; Drickamer et al., 1986; Kriegerand Herz, 1994; Elomaa et al., 1995) modulate the activity of LSR, bothas regards clearance of lipoproteins and as regards that of leptin.

Several proteins can interact with gC1q-R because they exhibithomologies with complement C1q. In particular, two proteins isolated inmice, AdipoQ (Hu et al., 1996) and acrp30 (Scherer et al., 1995), and ahuman protein APM1 (Maeda et al., 1996) exhibit marked homologies. Thesethree proteins, like the components of complement C1q (C1q A, B, C), aresecreted proteins; they have an NH₂-terminal end which resemblescollagen (repetition of Gly-X—Y motifs) and a COOH-terminal endcorresponding to the globular domain of complement C1q. These threeproteins are preferably expressed in the adipose tissue. There are only3 amino acids differing between AdipoQ and acrp30. APM1, a protein whosemessenger has been characterized as being highly expressed inadipocytes, exhibits 79.7% nucleic acid identity and 80.6% amino acididentity with AdipoQ. APM1 is therefore certainly the human homologue ofAdipoQ.

Example 13 Screening of Compounds Modifying the Activity of the LSRReceptor

As described above, the inventors formulated the hypothesis that the LSR“γ band”, a protein which is highly homologous to gC1qR, might interactwith the LSR receptor like a molecular chaperone and might thus form an“LSR complex”, comprising the α or α and β subunits of the LSR receptorand a gC1qR type molecule gC1qR has been previously identified as a cellsurface protein which binds the globular heads of the complement factorC1q. In addition to C1q, several proteins exhibiting homologies with theC1q proteins, in particular AdipoQ and acrp30 in mice and APM1 inhumans, are capable of interacting with the protein homologous to gC1qRin the LSR complex and of modifying the LSR activity.

Screening Parameters

The screening of a compound such as C1q or AdipoQ was carried outthrough the measurement of various parameters of which the mostimportant is the measurement of the effect of the compound on theactivity of the LSR receptor. The various parameters are the following:

-   -   change in weight    -   food intake    -   postprandial lipemic response    -   binding, internalization and/or degradation of lipoproteins such        as the LDLs.        Change in Weight

Osmotic pumps were surgically inserted into the abdominal cavities of 12Sprague-Dawley male rats of 400-450 g. The osmotic pumps containedeither 2 ml of PBS (phosphate buffered saline), pH 7.4 (control 6 rats),or 2 ml of recombinant AdipoQ protein (5 mg/ml PBS, 6 rats). These pumpswere designed to deliver 10 μl/h (50 μg AdipoQ/h). The animals areweighed and individually housed in metabolic cages. 3 animals in eachgroup are subjected ad libitum either to a normal diet or to a fattydiet (day 0). The fatty diet consists of a normal diet supplemented with2% (w/w) cholesterol, 10% (w/w) saturated fatty acid in the form ofvegetaline, [lacuna] % (w/w) sunflower oil and 15% (w/w) sucrose. On day3, the animals are weighed and blood samples are obtained from thecaudal vein. The quantity of plasma triglycerides was measured using anenzymatic kit.

Food Intake

Recombinant AdipoQ protein (100 μg) or PBS alone were injected daily for5 days through the caudal vein of ob/ob or db/db mice kept in ametabolic cage. The mice are weighed each day and the quantity of foodconsumed was also measured. The results correspond to a mean food intakeand a standard deviation for 4 mice in each group.

Postprandial Lipemic Response

Male Sprague-Dawley rats (400-450 g), starved since the day before, wereforce-fed with a meal which was very high in fat (t=0) (60% fatty acidof which 37% saturated, 27% monounsaturated and 36% polyunsaturated, 20%protein and 20% carbohydrate, the total providing 56 kcal/kg of bodyweight) and received immediately afterwards an intravenous injection(femoral vein) of either 300 μl of PBS alone or of the same volumecontaining 1 mg of mouse recombinant AdipoQ protein. Blood samples werecollected at various times (0, 2, 4 and 6 h). The quantity of plasmatriglycerides was measured using an enzymatic kit. The results arepresented as mean values and standard deviations on 3 animals.

LSR Activity or Binding, Internalization and Degradation of Lipoproteins

Primary cultures of rat hepatocytes were prepared and distributed into6-well plates (9000,000 cells/well). After 48 h, the cells were washedonce with PBS (2 ml/well) and incubated for 30 min at 37° C. with 20ng/ml of recombinant murine leptin. The cells were then incubated for 4h at 37° C. with increasing concentrations of recombinant murine AdipoQproteins and 20 μg/ml ¹²⁵I-LDL in the presence or in the absence of 0.5mM oleate. The binding, internalization and degradation of lipoproteinswere measured as indicated in Example 1.

C1q

The compound C1q was tested for its capacity to modulate the activity ofthe LSR receptor (binding, internalization and degradation oflipoproteins). FIG. 33 shows that the compound C1q exhibits the propertyof increasing the activity in the presence and in the absence of oleate.Thus, it was possible for this compound C1q to be selected as modulatorof the LSR activity through the test of activity described above.

AdipoQ

The compound AdipoQ was tested according to the four parameterspresented above.

FIG. 34 shows that the compound AdipoQ modulates the LSR activity in thepresence of oleate. Indeed, at the concentration of 25 ng/ml, itincreases the LSR activity.

FIG. 35 shows that the administration of AdipoQ makes it possible tomassively reduce the postprandial lipemic response.

FIG. 36 shows that a 3-day ip infusion treatment with AdipoQ causes aloss in weight which is much greater when the rat is subjected to afatty diet. Furthermore, the inventors observed that the level of plasmatriglycerides is reduced in the animals treated with AdipoQ.

FIG. 37 shows that an injection of AdipoQ reduces the food intake inobese animals.

The increase in the LSR activity induced by 25 ng/ml of AdipoQ canexplain the reduction in the postprandial lipemic response and theweight loss.

Thus, the AdipoQ protein is a very valuable compound which could be usedin particular in the treatment of obesity. The selection of this proteinas a candidate molecule in the treatment of obesity validates theparameters for screening a compound of interest modulating the LSRactivity, the most important parameter consisting in measuring the LSRactivity.

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1. A purified or recombinant Lipolysis Stimulated Receptor, wherein saidreceptor comprises a polypeptide having at least 90% homology to thepolypeptide of SEQ ID NO: 8 and wherein said polypeptide has at leastone biological activity selected from the group consisting of fatty acidbinding, clathrin binding leptin binding, and lipoprotein binding.
 2. Apurified or recombinant Lipolysis Stimulated Receptor that a) comprisesthe amino acid sequence of SEQ ID NO:8; or b) consists of the amino acidsequence of SEQ ID NO:8.
 3. The purified or recombinant receptor ofclaim 2, wherein said receptor comprises the amino acid sequence of SEQID NO:8.
 4. The polypeptide of claim 3, wherein said polypeptidecombines with one or more heterologous polypeptides to form an LSRreceptor complex, and wherein said complex comprises an α subunit or anα′ subunit, and at least one β subunit.
 5. The polypeptide of claim 4,wherein said complex comprises three β subunits.
 6. The polypeptide ofclaim 4, wherein said polypeptide is from a human, and wherein saidpolypeptide has a molecular weight of 64 kD.
 7. The polypeptide of claim4, wherein said polypeptide is expressed in hepatic cells.
 8. Thepolypeptide of claim 4, wherein said complex has a biological activityselected from the group consisting of lipoprotein binding, lipoproteininternalization, and lipoprotein degradation.
 9. The polypeptide ofclaim 4, wherein said complex has a biological activity that is selectedfrom the group consisting of leptin binding, leptin internalization, andleptin degradation.
 10. A composition comprising the polypeptide ofclaim
 3. 11. The composition of claim 10, further comprising aphysiologically acceptable carrier.
 12. The purified or recombinantreceptor of claim 2, wherein said receptor consists of the amino acidsequence of SEQ ID NO:8.
 13. An isolated or recombinant biologicallyactive polypeptide fragment of SEQ ID NO: 8, said fragment comprising anamino acid sequence selected from the group consisting of: a) an aminoacid sequence spanning amino acids 76 to 94 of SEQ ID NO:8; b) an aminoacid sequence spanning amino acids 76 to 160 of SEQ ID NO:8; c) an aminoacid sequence spanning amino acids 76 to 237 of SEQ ID NO:8; d) an aminoacid sequence spanning amino acids 157 to 249 of SEQ ID NO:8; e) anamino acid sequence spanning amino acids 236 to 530 of SEQ ID NO:8; f)an amino acid sequence spanning amino acids 236 to 613 of SEQ ID NO: 8;and g) an amino acid sequence spanning amino acids 76 to 613 of SEQ IDNO:8.
 14. The polypeptide fragment of claim 13, wherein said polypeptidecombines with one or more heterologous polypeptides to form an LSRreceptor complex, and wherein said complex comprises an α subunit or anα′ subunit, and at least one β subunit.
 15. The polypeptide fragment ofclaim 14, wherein said complex comprises three β subunits.
 16. Thepolypeptide fragment of claim 14, wherein said polypeptide is from ahuman, and wherein said polypeptide has a molecular weight of 64 kD. 17.The polypeptide fragment of claim 14, wherein said polypeptide isexpressed in hepatic cells.
 18. The polypeptide fragment of claim 14,wherein said complex has a biological activity selected from the groupconsisting of lipoprotein binding, lipoprotein internalization, andlipoprotein degradation.
 19. The polypeptide fragment of claim 14,wherein said complex has a biological activity that is selected from thegroup consisting of leptin binding, leptin internalization, and leptindegradation.
 20. The polypeptide fragment of claim 13, wherein saidpolypeptide is recombinant.
 21. A composition comprising the polypeptideof claim
 13. 22. The composition of claim 21, further comprising aphysiologically acceptable carrier.
 23. The isolated or recombinantbiologically active polypeptide fragment of claim 13, said fragmentcomprising an amino acid sequence selected from the group consisting of:a) an amino acid sequence spanning amino acids 76 to 94 of SEQ ID NO:8that contains a fatty acid binding site; b) an amino acid sequencespanning amino acids 76 to 160 of SEQ ID NO:8 that contains a fatty acidbinding site and a clathrin binding site; c) an amino acid sequencespanning amino acids 76 to 237 of SEQ ID NO:8 that contains a fatty acidbinding site, a clathrin binding site and contains a transport signal;d) an amino acid sequence spanning amino acids 157 to 249 of SEQ ID NO:8that contains a clathrin binding site and contains a transport signal;e) an amino acid sequence spanning amino acids 236 to 530 of SEQ ID NO:8and that contains a transport signal, a leptin binding site and a RSRSmotif; f) an amino acid sequence spanning amino acids 236 to 613 of SEQID NO: 8 and that contains a transport signal, a leptin binding site, aRSRS motif, and a lipoprotein binding site; and g) an amino acidsequence spanning amino acids 76 to 613 of SEQ ID NO:8 and that containsa fatty acid binding site, a clathrin binding site, contains a transportsignal, contains a leptin binding site, contains an RSRS motif, and hasa lipoprotein binding site.